U.S. patent application number 10/807807 was filed with the patent office on 2005-08-18 for high-throughput diagnostic assay for the human virus causing severe acute respiratory syndrome (sars).
Invention is credited to Chan, Kwok Hung, Guan, Yi, Leung, Frederick C., Nicholls, John M., Peiris, Joseph S.M., Poon, Lit Man, Yuen, Kwok Yung.
Application Number | 20050181357 10/807807 |
Document ID | / |
Family ID | 33102680 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181357 |
Kind Code |
A1 |
Peiris, Joseph S.M. ; et
al. |
August 18, 2005 |
High-throughput diagnostic assay for the human virus causing severe
acute respiratory syndrome (SARS)
Abstract
The present invention relates to a high-throughput diagnostic
assay for the virus causing Severe Acute Respiratory Syndrome
(SARS) in humans ("hSARS virus"). In particular, the invention
relates to a high-throughput reverse transcription-PCR diagnostic
test for SARS associated coronavirus (SARS-CoV). The present assay
is a rapid, reliable assay which can be used for diagnosis and
monitoring the spread of SARS and is based on the nucleotide
sequences of the N (nucleocapsid)-gene of the hSARS virus. The
present method eliminates false negative results and provides
increased sensitivity for the assay. The invention also discloses
the S (spike)-gene of the hSARS virus. The invention further
relates to the deduced amino acid sequences of the N-gene and
S-gene products of the hSARS virus and to the use of the N-gene and
S-gene products in diagnostic methods. The invention further
encompasses diagnostic assays and kits comprising antibodies
generated against the N-gene or S-gene product.
Inventors: |
Peiris, Joseph S.M.; (Hong
Kong, CN) ; Yuen, Kwok Yung; (Hong Kong, CN) ;
Poon, Lit Man; (Hong Kong, CN) ; Guan, Yi;
(Honk Kong, CN) ; Chan, Kwok Hung; (Hong Kong,
CN) ; Nicholls, John M.; (Hong Kong, CN) ;
Leung, Frederick C.; (Hong Kong, CN) |
Correspondence
Address: |
DICKSTEIN SHAPIRO MORIN & OSHINSKY LLP
1177 AVENUE OF THE AMERICAS (6TH AVENUE)
41 ST FL.
NEW YORK
NY
10036-2714
US
|
Family ID: |
33102680 |
Appl. No.: |
10/807807 |
Filed: |
March 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60457031 |
Mar 24, 2003 |
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60457730 |
Mar 26, 2003 |
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60459931 |
Apr 2, 2003 |
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60460357 |
Apr 3, 2003 |
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60461265 |
Apr 8, 2003 |
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60462805 |
Apr 14, 2003 |
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60464886 |
Apr 23, 2003 |
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60465738 |
Apr 25, 2003 |
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60470935 |
May 14, 2003 |
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Current U.S.
Class: |
435/5 ; 530/350;
530/388.3; 536/23.72 |
Current CPC
Class: |
Y02A 50/451 20180101;
C12Q 1/701 20130101; C12Q 1/6844 20130101; A61P 11/00 20180101;
A61P 31/14 20180101; Y02A 50/30 20180101 |
Class at
Publication: |
435/005 ;
530/388.3; 536/023.72; 530/350 |
International
Class: |
C12Q 001/70; C07H
021/02; C07K 014/165; C07K 016/10 |
Claims
What is claimed:
1. An isolated nucleic acid molecule consisting essentially of a
nucleic acid sequence of SEQ ID NO:2471, or a complement
thereof.
2. An isolated nucleic acid molecule consisting essentially of a
nucleic acid sequence of SEQ ID NO:2473, or a complement
thereof.
3. An isolated nucleic acid molecule which hybridizes under
stringent conditions to a nucleic acid molecule having the
nucleotide sequence of claim 1 or 2, or a complement thereof.
4. The nucleic acid molecule of claim 1 or 2 wherein the molecule
is RNA.
5. The nucleic acid molecule of claim 1 or 2 wherein the molecule
is DNA.
6. An isolated nucleic acid molecule which hybridizes under
stringent conditions to the nucleic acid molecule of claim 1 or 2,
or a complement thereof, wherein the nucleic acid molecule encodes
an amino acid sequence which has a biological activity exhibited by
a polypeptide encoded by the nucleotide sequence of SEQ ID NO:2471
or 2473.
7. An isolated polypeptide encoded by the nucleic acid molecule of
claim 1 or 2.
8. An antibody or an antigen-binding fragment thereof which
immunospecifically binds to the N-gene protein of a hSARS
virus.
9. An antibody or an antigen-binding fragment thereof which
immunospecifically binds to the S-gene protein of a hSARS
virus.
10. The antibody of claim 8, 9, or an antigen-binding fragment
thereof which neutralizes the hSARS virus.
11. An antibody which immunospecifically binds to the polypeptide
of claim 7, or an antigen-binding fragment of said antibody.
12. A method for detecting the presence of a N-gene of the hSARS
virus in a biological sample, said method comprising: (a)
contacting the sample with a compound that selectively binds to
said N-gene; and (b) detecting whether the compound binds to said
N-gene in the sample.
13. The method of claim 12, wherein the compound that binds to said
N-gene is a nucleic acid molecule comprising a nucleotide sequence
having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150 or 1,200
contiguous nucleotides of the nucleotide sequence of SEQ ID
NO:2471, or a complement thereof.
14. The method of claim 12, wherein the compound that binds to said
N-gene is a nucleic acid molecule comprising a nucleotide sequence
of SEQ ID NO:2475, 2476, 2480 and/or 2481.
15. A method for detecting the presence of a S-gene of the hSARS
virus in a biological sample, said method comprising: (a)
contacting the sample with a compound that selectively binds to
said S-gene; and (b) detecting whether the compound binds to said
S-gene in the sample.
16. The method of claim 15, wherein the compound that binds to said
S-gene is a nucleic acid molecule comprising a nucleotide sequence
having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200,
2,000, or 3,000 contiguous nucleotides of the nucleotide sequence
of SEQ ID NO:2473, or a complement thereof.
17. The method of claim 15, wherein the compound that binds to said
S-gene is a nucleic acid molecule comprising a nucleotide sequence
of SEQ ID NO:2477 and/or 2478.
18. A method for detecting the presence of the polypeptide of claim
7 in a sample, said method comprising: (a) contacting the sample
with a compound that selectively binds to said polypeptide; and (b)
detecting whether the compound binds to said polypeptide in the
sample.
19. The method of claim 18, wherein the compound that binds to the
polypeptide is an antibody.
20. A method for identifying a subject infected with the hSARS
virus, said method comprising: (a) obtaining total RNA from a
biological sample obtained from the subject (b) reverse
transcribing the total RNA to obtain cDNA; and (c) subjecting the
cDNA to real-time PCR assay using a set of primers derived from a
nucleotide sequence of the N-gene of the hSARS.
21. The method of claim 20, wherein the set of primers have
nucleotide sequences of SEQ ID NOS:2475 and 2476, respectively.
22. The method of claim 20, wherein the set of primers have
nucleotide sequences of SEQ ID NOS:2480 and 2481, respectively.
23. A method for identifying a subject infected with the hSARS
virus, said method comprising: (a) obtaining total RNA from a
biological sample obtained from the subject (b) reverse
transcribing the total RNA to obtain cDNA; and (c) subjecting the
cDNA to real-time PCR assay using a set of primers derived from a
nucleotide sequence of the S-gene of the hSARS.
24. The method of claim 23, wherein the set of primers have
nucleotide sequences of SEQ ID NOS:2477 and 2478, respectively.
25. A kit comprising in one or more containers one or more isolated
nucleic acid molecules comprising a nucleotide sequence of SEQ ID
NO:2475 and/or SEQ ID NO:2476.
26. A kit comprising in one or more containers one or more isolated
nucleic acid molecules comprising a nucleotide sequence of SEQ ID
NO:2480 and/or SEQ ID NO:2481.
27. A kit comprising in one or more containers one or more isolated
nucleic acid molecules comprising a nucleotide sequence of SEQ ID
NO:2477 and/or SEQ ID NO:2478.
28. A kit comprising in one or more containers one or more
antibodies of claim 8 or 9.
29. A kit comprising in one or more containers one or more
antibodies of claim 11.
Description
[0001] This application claims priority benefit to U.S. provisional
application No. 60/457,031, filed Mar. 24, 2003; U.S. provisional
application No. 60/457,730, filed Mar. 26, 2003; U.S. provisional
application No. 60/459,931, filed Apr. 2, 2003; U.S. provisional
application No. 60/460,357, filed Apr. 3, 2003; U.S. provisional
application No. 60/461,265, filed Apr. 8, 2003; U.S. provisional
application No. 60/462,805, filed Apr. 14, 2003; U.S. provisional
application No. 60/464,886 filed Apr. 23, 2003, U.S. provisional
application No. 60/465,738, filed Apr. 25, 2003; and U.S.
provisional application No. 60/470,935, filed May 14, 2003, each of
which is incorporated herein by reference in its entirety.
[0002] The instant application contains a lengthy Sequence listing
which is being concurrently submitted via triplicate CD-R in lieu
of a printed paper copy, and is hereby inconrporated by reference
in its entirety. Said CD-R, recorded on Mar. 22, 2004, are labeled
"CRF", "Copy 1" and "Copy 2", respectively, and each contains only
identical 1.6 MB file (V9661077.APP).
1. INTRODUCTION
[0003] The present invention relates to a high-throughput
diagnostic assay for the virus causing Severe Acute Respiratory
Syndrome (SARS) in humans ("hSARS virus"). In particular, the
invention relates to a high-throughput reverse transcription-PCR
diagnostic test for SARS associated coronavirus (SARS-CoV). The
present assay is a rapid, reliable assay which may be used for
diagnosis and monitoring the spread of SARS. The present method
eliminates false negative results and provides increased
sensitivity for the assay. The invention further relates to
nucleotide sequences and portions thereof, useful for the diagnosis
of SARS. The invention further relates to nucleotide sequences and
portions thereof, useful for assessing genetic diversity of SARS.
The nucleotide sequences of the present invention comprise the
(Nucleocapsid) N-gene and the S-gene sequences of the hSARS virus.
The invention relates to a diagnostic kit that comprises nucleic
acid molecules for the detection of the N-gene or S-gene of hSARS
virus. The invention also relates to the deduced amino acid
sequences of the N-gene and S-gene products of the hSARS virus. The
invention further relates to the use of the N-gene and S-gene
products in diagnostic methods. The invention further encompasses
diagnostic assays and kits comprising antibodies generated against
the N-gene or S-gene product.
2. BACKGROUND OF THE INVENTION
[0004] Recently, there has been an outbreak of atypical pneumonia
in Guangdong province in mainland China. Between November 2002 and
March 2003, there were 792 reported cases with 31 fatalities (WHO.
Severe Acute Respiratory Syndrome (SARS) Weekly Epidemiol Rec.
2003; 78: 86). Patients with SARS show various clinical symptoms,
including fever (of 38 degrees Celsius or above for over 24 hours),
malaise, chills, headache and body ache. Chest X-rays show changes
compatible with pneumonia. Other symptoms include coughing,
shortness of breath or difficulty in breathing. By 3 May 2003, a
cumulative total number of 1621 cases and 179 deaths had been
occurred in Hong Kong, which contributed to 26% and 41% of the
global reported cases (6234) and deaths (435) respectively. As the
disease is highly contagious and spreads in daily-life activities,
it is important to develop a rapid and reliable diagnosis test to
monitor and control the disease. In response to this crisis, the
Hospital Authority in Hong Kong has increased the surveillance on
patients with severe atypical pneumonia. In the course of this
investigation, a number of clusters of health care workers with the
disease were identified. In addition, there were clusters of
pneumonia incidents among persons in close contact with those
infected. The disease was unusual in its severity and its
progression in spite of the antibiotic treatment typical for the
bacterial pathogens that are known to be commonly associated with
atypical pneumonia. The present inventors were one of the groups
involved in the investigation of these patients. All tests for
identifying commonly recognized viruses and bacteria were negative
in these patients. Furthermore, diagnostic tests for the detection
of other genes in the hSARS virus, such as the 1b-gene are not
useful to accurately diagnose SARS. The disease was given the
acronym Severe Acute Respiratory Syndrome ("SARS"). This virus
mutates and changes rapidly and hence the diagnostic of SARS was
extremely difficult until the isolation of particular regions of
the virus, the N-gene and S-gene, of the hSARS virus from the SARS
patients by the present inventors as disclosed herein. Namely, the
present invention discloses a diagnostic assay using particular
regions in the genome of the virus for rapid, accurate, reliable
and specific identification of the hSARS virus. The invention is
useful in both clinical and scientific research applications.
Furthermore, the present invention provides a high-throughput assay
which can be used as a sensitive method for diagnosis and
monitoring the spread of the SARS.
3. SUMMARY OF INVENTION
[0005] The present invention is based upon the inventors'
identification of a specific region of the hSARS virus,
specifically, the 3'region of the hSARS viral genome, and in
particular, the (nucleocapsid) N-gene of the hSARS virus that may
be used in diagnostic assay to detect hSARS. In particular, the
N-gene is useful for the diagnosis of SARS because the N-gene has
the most abundant copy number during viral infection compared to
any other gene in the hSARS virus, especially when the cells are
lysed. Thus, the nucleic acid sequences of the N-gene of the hSARS
virus are particularly useful in a rapid and reliable diagnostic
assay for the hSARS virus. Furthermore, the present method
eliminates false negative results and increases the sensitivity of
the assay.
[0006] The hSARS virus was isolated from patients suffering from
SARS in the recent outbreak of severe atypical pneumonia in China.
The isolated virus is an enveloped, single-stranded RNA virus of
positive polarity which belongs to the order, Nidovirales, of the
family, Coronaviridae. The hSARS virus is a very large RNA virus
consisting of approximately 29,742 nucleotides. The complete
genomic sequence of the hSARS virus was deposited in Genbank, NCBI
with Accession No: AY278491 (SEQ ID NO:15), which is incorporated
by reference. The isolated hSARS virus was deposited with China
Center for Type Culture Collection (CCTCC) on Apr. 2, 2003 and
accorded an accession number, CCTCC-V200303, as described in
Section 7, infra, which is incorporated by reference. Also, the
entire genome sequence of the hSARS virus, CCTCC-V200303, and
characterization thereof are disclosed in a United States patent
application with Attorney Docket No. V9661.0069 filed concurrently
herewith on Mar. 24, 2004, which is incorporated by reference in
its entirety. The virus mutates and changes rapidly and hence
making the diagnostic of SARS very difficult. The present inventors
have designed a diagnostic assay for detecting the presence of
N-gene nucleic acid sequence or protein to rapidly, accurately, and
specifically identify the hSARS virus. Furthermore, the present
inventors have designed a diagnostic assay for detecting the
presence of S-gene nucleic acid sequence or protein to determine
the genetic diversity of the hSARS virus. Accordingly, the
invention relates to methods of detecting nucleotide sequences of
the N-gene and S-gene of the hSARS virus.
[0007] The present invention provides a rapid, reliable assay for
the detection of hSARS virus. In preferred embodiment, the
detection of hSARS virus includes the use of the nucleic acid
molecules of the present invention in a polymerase chain reaction,
Reverse transcription-Polymeras- e Chain Reaction (RT-PCR),
Southern analysis, Northern analysis, or other nucleic acid
hybridization for the detection of hSARS nucleic acids. In one
embodiment, the invention provides methods for detecting the
presence, activity or expression of the hSARS virus of the
invention in a biological material, such as cells, nasopharyngeal
aspirate, sputum, blood, saliva, urine, stool and so forth. In
preferred embodiments, the biological material is nasopharyngeal
aspirate or stool. The increased or decreased activity or
expression of the hSARS virus in a sample relative to a control
sample can be determined by contacting the biological material with
an agent which can detect directly or indirectly the presence,
activity or expression of the hSARS virus. In a specific
embodiment, the detecting agents are the nucleic acid molecules of
the present invention.
[0008] The present invention also relates to a method for
identifying a subject infected with the hSARS virus, said method
comprising obtaining total RNA from a biological sample obtained
from the subject; reverse transcribing the total RNA to obtain
cDNA; and subjecting the cDNA to PCR assay using a set of primers
derived from a nucleotide sequence of the hSARS. In preferred
embodiments, the primers are derived from the (nucleocapid) N-gene.
In most preferred embodiments, the primers comprise the nucleotide
sequences of SEQ ID NOS:2475 and/or 2476 or SEQ ID NOS:2480 and/or
2481. In another preferred embodiments, the primers are derived
from the (spike) S-gene. In more preferred embodiments, the primers
comprise the nucleotide sequences of SEQ ID NOS:2477 and/or
2478.
[0009] The invention further relates to the use of the sequence
information of the isolated virus for diagnostic and therapeutic
methods. In a specific embodiment, the invention provides nucleic
acid molecules which are suitable for use as primers consisting of
or comprising the nucleotide sequence of SEQ ID NO:1, 11, 13, 15,
2471, or 2473, or a complement thereof, or at least a portion of
the nucleotide sequence thereof. In the most preferred embodiment,
the primers comprise the nucleic acid sequence of SEQ ID NOS:2475
and/or 2476 or SEQ ID NOS:2480 and/or 2481 for the detection of
N-gene. In another most preferred embodiment, the primers comprise
the nucleic acid sequence of SEQ ID NO:2477 and/or 2478 for the
detection of S-gene. In another specific embodiment, the invention
provides nucleic acid molecules which are suitable for
hybridization to hSARS nucleic acid, including, but not limited to,
as PCR primers, Reverse Transcriptase primers, probes for Southern
analysis or other nucleic acid hybridization analysis for the
detection of hSARS nucleic acids, e.g., consisting of or comprising
the nucleotide sequence of SEQ ID NO:1, 11, 13, 15, 2471, 2473,
2475, 2476, 2477, 2478, 2480 or 2481, or a complement thereof, or a
portion thereof. In a preferred embodiment, primers that amplify
fragments comprising (nucleotide position 18057 to 18222 or
portions thereof of SEQ ID NO:15) 1b gene; (nucleotide position
21920-22107, or portions thereof of SEQ ID NO:15) M-gene; and
(nucleotide position 28658-28883, or portions thereof of SEQ ID
NO:15) N-gene may be used for probe synthesis for detection of
hSARS nucleic acids. In a specific embodiment, the invention
provides a diagnostic kit comprising nucleic acid molecules which
are suitable for use to detect the N-gene of hSARS. In a specific
embodiment, the N-gene comprises nucleic acid sequence of SEQ ID
NO: 2471. In specific embodiment, the nucleic acid molecules
comprise nucleic acid sequence of SEQ ID NOS:2475 and/or 2476 or
SEQ ID NOS:2480 and/or 2481. In preferred embodiments, the
diagnostic kit further comprises a control for amplification of 1b
gene. In specific embodiments, the primers used for amplifying 1 b
gene are SEQ ID NOS:3 and/or 4. In another specific embodiments,
the diagnostic kit further comprises an internal control using pig
.beta.-actin gene. In specific embodiments, the primers used for
amplifying .beta.-actin gene are SEQ ID NOS:2482 and/or 2483.
[0010] In a specific embodiment, the invention provides a
diagnostic kit comprising nucleic acid molecules which are suitable
for use to detect the S-gene of hSARS. In a specific embodiment,
the S-gene comprises nucleic acid sequence of SEQ ID NO: 2473. In
specific embodiment, the nucleic acid molecules comprise nucleic
acid sequence of SEQ ID NOS: 2477 and/or 2478. The invention
further encompasses chimeric or recombinant viruses encoded in
whole or in part by said nucleotide sequences.
[0011] In another specific embodiment, the invention provides
nucleic acid molecules comprising a nucleotide sequence of SEQ ID
NO:1, 11, 13, 2471, and/or 2473. In a specific embodiment, the
present invention provides isolated nucleic acid molecules
comprising or, alternatively, consisting of the nucleotide sequence
of SEQ ID NO:1, a complement thereof or a portion thereof,
preferably at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,
200, 300, 350, 400, 450, 500, 550, 600, or more contiguous
nucleotides of the nucleotide sequence of SEQ ID NO:1, or a
complement thereof. In another specific embodiment, the present
invention provides isolated nucleic acid molecules comprising or,
alternatively, consisting of the nucleotide sequence of SEQ ID
NO:11, a complement thereof or a portion thereof, preferably at
least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
1,050, 1,100, 1,150, 1,200, or more contiguous nucleotides of the
nucleotide sequence of SEQ ID NO:11, or a complement thereof. In
yet another specific embodiment, the present invention provides
isolated nucleic acid molecules comprising or, alternatively,
consisting of the nucleotide sequence of SEQ ID NO:13, a complement
thereof or a portion thereof, preferably at least 5, 10, 15, 20,
25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550,
600, 650, 700, or more contiguous nucleotides of the nucleotide
sequence of SEQ ID NO:13, or a complement thereof. In another
specific embodiment, the present invention provides isolated
nucleic acid molecules comprising or, alternatively, consisting of
the nucleotide sequence of SEQ ID NO:15, a complement thereof or a
portion thereof, preferably at least 5, 10, 15, 20, 25, 30, 35, 40,
45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000,
3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000,
12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000,
20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000,
28,000, 29,000 or more contiguous nucleotides of the nucleotide
sequence of SEQ ID NO:15, or a complement thereof. In another
specific embodiment, the present invention provides isolated
nucleic acid molecules comprising or, alternatively, consisting of
the nucleotide sequence of SEQ ID NO:2471, a complement thereof or
a portion thereof, preferably at least 5, 10, 15, 20, 25, 30, 35,
40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200 or more
contiguous nucleotides of the nucleotide sequence of SEQ ID
NO:2471, or a complement thereof. In another specific embodiment,
the present invention provides isolated nucleic acid molecules
comprising or, alternatively, consisting of the nucleotide sequence
of SEQ ID NO:2473, a complement thereof or a portion thereof,
preferably at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,
200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, or more
contiguous nucleotides of the nucleotide sequence of SEQ ID
NO:2473, or a complement thereof. Furthermore, in another specific
embodiment, the invention provides isolated nucleic acid molecules
which hybridize under stringent conditions, as defined herein, to a
nucleic acid molecule having the sequence of SEQ ID NO:1, 11, 13,
15, 2471, or 2473, or a complement thereof. In one embodiment, the
invention provides an isolated nucleic acid molecule which is
antisense to the coding strand of a nucleic acid of the invention.
In another specific embodiment, the invention provides isolated
polypeptides or proteins that are encoded by a nucleic acid
molecule comprising or, alternatively consisting of a nucleotide
sequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100,
150, 200, 300, 350, 400, 450, 500, 550, 600, or more contiguous
nucleotides of the nucleotide sequence of SEQ ID NO:1, or a
complement thereof. In yet another specific embodiment, the
invention provides isolated polypeptides or proteins that are
encoded by a nucleic acid molecule comprising or, alternatively
consisting of a nucleotide sequence that is at least 5, 10, 15, 20,
25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,
1,200 or more contiguous nucleotides of the nucleotide sequence of
SEQ ID NO:11, or a complement thereof. In yet another specific
embodiment, the invention provides isolated polypeptides or
proteins that are encoded by a nucleic acid molecule comprising or,
alternatively consisting of a nucleotide sequence that is at least
5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, or more contiguous nucleotides of the
nucleotide sequence of SEQ ID NO:13, or a complement thereof. In
yet another specific embodiment, the invention provides isolated
polypeptides or proteins that are encoded by a nucleic acid
molecule comprising or, alternatively consisting of a nucleotide
sequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100,
150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000,
4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,
13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000,
21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000,
29,000 or more contiguous nucleotides of the nucleotide sequence of
SEQ ID NO:15, or a complement thereof. In yet another specific
embodiment, the invention provides isolated polypeptides or
proteins that are encoded by a nucleic acid molecule comprising or,
alternatively consisting of a nucleotide sequence that is at least
5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
1,050, 1,100, 1,150, 1,200 or more contiguous nucleotides of the
nucleotide sequence of SEQ ID NO:2471, or a complement thereof. In
yet another specific embodiment, the invention provides isolated
polypeptides or proteins that are encoded by a nucleic acid
molecule comprising or, alternatively consisting of a nucleotide
sequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100,
150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, or
more contiguous nucleotides of the nucleotide sequence of SEQ ID
NO:2473, or a complement thereof. The invention further provides
proteins or polypeptides that are isolated from the hSARS virus,
including viral proteins isolated from cells infected with the
virus but not present in comparable uninfected cells. The invention
further provides proteins or polypeptides shown in FIGS. 11 (SEQ ID
NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589,
1591-1964 and 1966-2470). The invention further provides proteins
or polypeptides having the amino acid sequence of SEQ ID NO:2472 or
2474. The polypeptides or the proteins of the present invention
preferably have a biological activity of the protein (including
antigenicity and/or immunogenicity) encoded by the sequence of SEQ
ID NO:1, 11, 13, 2471, or 2473. In other embodiments, the
polypeptides or the proteins of the present invention have a
biological acitivity of the protein (including antigenicity and/or
immunogenicity) encoded by a nucleotide sequence that is at least
5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000,
15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000,
23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more
contiguous nucleotides of the nucleotide sequence of SEQ ID NO:15,
or a complement thereof. In other embodiments, the polypeptides or
the proteins of the present invention have a biological activity of
the protein (including antigenicity and/or immunogenicity) of FIGS.
11 (SEQ ID NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID
NOS:1109-1589, 1591-1964 and 1966-2470). The invention further
provides proteins or polypeptides having a biological activity of
the protein having amino acid sequence of SEQ ID NO: 2472 or
2474.
[0012] In one aspect, the invention provides a method for
propagating the hSARS virus in host cells comprising infecting the
host cells with the isolated hSARS virus, culturing the host cells
to allow the virus to multiply, and harvesting the resulting
virions. Also provide by the present invention are host cells that
are infected with the hSARS virus.
[0013] In one aspect, the invention relates to the use of the
isolated hSARS virus for diagnostic and therapeutic methods. In a
specific embodiment, the invention provides a method of detecting
in a biological sample an antibody immunospecific for the hSARS
virus using the isolated hSARS virus or any proteins or
polypeptides thereof. In another specific embodiment, the invention
provides a method of screening for an antibody which
immunospecifically binds and neutralizes hSARS. Such an antibody is
useful for a passive immunization or immunotherapy of a subject
infected with hSARS.
[0014] The invention further provides antibodies that specifically
bind a polypeptide of the invention encoded by the nucleotide
sequence of SEQ ID NO:1, 11, 13, 2471, or 2473, or a fragment
thereof, or encoded by a nucleic acid comprising a nucleotide
sequence that hybridizes under stringent conditions to the
nucleotide sequence of SEQ ID NO:1, 11, 13, 2471, or 2473, and/or
any hSARS epitope, having one or more biological activities of a
polypeptide of the invention. The invention further provides
antibodies that specifically bind polypeptides of the invention
encoded by the nucleotide sequence of SEQ ID NO:15, or a fragment
thereof. These polypeptides include those shown in FIGS. 11 (SEQ ID
NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589,
1591-1964 and 1966-2470). In another embodiment, the polypeptide
comprises amino acid sequence of SEQ ID NO:2472, or 2474. The
invention further provides antibodies that specifically bind
polypeptides of the invention encoded by a nucleic acid comprising
a nucleotide sequence that hybridizes under stringent conditions to
the nucleotide sequence of SEQ ID NO:15, and/or any hSARS epitope,
having one or more biological activities of a polypeptide of the
invention. Such antibodies include, but are not limited to
polyclonal, monoclonal, bi-specific, multi-specific, human,
humanized, chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, disulfide-linked Fvs,
intrabodies and fragments containing either a Vl or VH domain or
even a complementary determining region (CDR) that specifically
binds to a polypeptide of the invention.
[0015] In another embodiment, the invention provides vaccine
preparations, comprising the hSARS virus, including recombinant and
chimeric forms of said virus, or protein subunits of the virus. In
a specific embodiment, the vaccine preparations of the present
invention comprise live but attenuated hSARS virus with or without
adjuvants. In another specific embodiment, the vaccine preparations
of the invention comprise an inactivated or killed hSARS virus.
Such attenuated or inactivated viruses may be prepared by a series
of passages of the virus through the host cells or by preparing
recombinant or chimeric forms of virus. Accordingly, the present
invention further provides methods of preparing recombinant or
chimeric forms of hSARS. In another specific invention, the vaccine
preparations of the present invention comprise a nucleic acid or
fragment of the hSARS virus, e.g., the virus having accession no.
CCTCC-V200303, or nucleic acid molecules having the sequence of SEQ
ID NO. 1, 11, 13, 15, 2471 or 2473, or a fragment thereof. In
another embodiment, the invention provides vaccine preparations
comprising one or more polypeptides isolated from or produced from
nucleic acid of hSARS virus, for example, of deposit accession no.
CCTCC-V200303. In a specific embodiment, the vaccine preparations
comprise a polypeptide of the invention encoded by the nucleotide
sequence of SEQ ID NO:1, 11, 13, 2471 or 2473, or a fragment
thereof. In a specific embodiment, the vaccine preparations
comprise polypeptides of the invention as shown in FIGS. 11 (SEQ ID
NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NO:1109-1589,
1591-1964 AND 1966-2470) or encoded by the nucleotide sequence of
SEQ ID NO:15, or a fragment thereof. In a specific embodiment, the
vaccine preparations comprise polypeptides comprising amino acid
sequence of SEQ ID NO:2472 or 2474. Furthermore, the present
invention provides methods for treating, ameliorating, managing or
preventing SARS by administering the vaccine preparations or
antibodies of the present invention alone or in combination with
adjuvants, or other pharmaceutically acceptable excipients.
[0016] In another aspect, the present invention provides
pharmaceutical compositions comprising anti-viral agents of the
present invention and a pharmaceutically acceptable carrier. In a
specific embodiment, the anti-viral agent of the invention is an
antibody that immunospecifically binds hSARS virus or any hSARS
epitope. In preferred embodiments, such antibodies neutralize the
hSARS virus. In a specific embodiment, the anti-viral agent of the
invention binds a fragment, variant, homolog of N-gene or S-gene of
hSARS virus. In a specific embodiment, the anti-viral agent of the
invention binds a fragment, variant, homolog of a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2472 or 2474. In
another specific embodiment, the anti-viral agent is a polypeptide
or protein of the present invention or nucleic acid molecule of the
invention. The invention also provides kits containing a
pharmaceutical composition of the present invention.
[0017] 3.1 Definitions
[0018] The term "an antibody or an antibody fragment that
immunospecifically binds a polypeptide of the invention" as used
herein refers to an antibody or a fragment thereof that
immunospecifically binds to the polypeptide encoded by the
nucleotide sequence of SEQ ID NO:1, 11, 13, 15, 2471 2473, or the
polypeptides shown in FIGS. 11 and 12, or a fragment thereof, and
does not non-specifically bind to other polypeptides. An antibody
or a fragment thereof that immunospecifically binds to the
polypeptide of the invention may cross-react with other antigens.
Preferably, an antibody or a fragment thereof that
immunospecifically binds to a polypeptide of the invention does not
cross-react with other antigens. An antibody or a fragment thereof
that immunospecifically binds to the polypeptide of the invention,
can be identified by, for example, immunoassays or other techniques
known to those skilled in the art.
[0019] An "isolated" or "purified" peptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free of chemical precursors or other
chemicals when chemically synthesized. The language "substantially
free of cellular material" includes preparations of a
polypeptide/protein in which the polypeptide/protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. Thus, a polypeptide/protein that is
substantially free of cellular material includes preparations of
the polypeptide/protein having less than about 30%, 20%, 10%, 5%,
2.5%, or 1%, (by dry weight) of contaminating protein. When the
polypeptide/protein is recombinantly produced, it is also
preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, 10%, or 5% of the volume of
the protein preparation. When polypeptide/protein is produced by
chemical synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. Accordingly, such preparations of the
polypeptide/protein have less than about 30%, 20%, 10%, 5% (by dry
weight) of chemical precursors or compounds other than
polypeptide/protein fragment of interest. In a preferred embodiment
of the present invention, polypeptides/proteins are isolated or
purified.
[0020] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In a preferred embodiment of the invention, nucleic acid molecules
encoding polypeptides/proteins of the invention are isolated or
purified. The term "isolated" nucleic acid molecule does not
include a nucleic acid that is a member of a library that has not
been purified away from other library clones containing other
nucleic acid molecules.
[0021] The term "portion" or "fragment" as used herein refers to a
fragment of a nucleic acid molecule containing at least about 25,
30, 35, 40, 45, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,
8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000,
16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000,
24,000, 25,000, 26,000, 27,000, 28,000, 29,000, or more contiguous
nucleic acids in length of the relevant nucleic acid molecule and
having at least one functional feature of the nucleic acid molecule
(or the encoded protein has one functional feature of the protein
encoded by the nucleic acid molecule); or a fragment of a protein
or a polypeptide containing at least 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 120, 140, 160, 180, 200,
220, 240, 260, 280, 300, 320, 340, 360, 400, 500, 600, 700, 800,
900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,100, 4,200,
4,300, 4,350, 4,360, 4,370, 4,380 amino acid residues in length of
the relevant protein or polypeptide and having at least one
functional feature of the protein or polypeptide.
[0022] The term "3' region of the hSAR viral genome" refers to from
about nucleotide position 18,000 to 29742 of SEQ ID NO:15.
[0023] The term "having a biological activity of the protein" or
"having biological activities of the polypeptides of the invention"
refers to the characteristics of the polypeptides or proteins
having a common biological activity similar or identical structural
domain and/or having sufficient amino acid identity to the
polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 11,
13, 15, 16, 240, 737, 1108, 1590, 1965, 2471 or 2473. Such common
biological activities of the polypeptides of the invention include
antigenicity and immunogenicity.
[0024] The term "under stringent condition" refers to hybridization
and washing conditions under which nucleotide sequences having at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% identity to each other remain hybridized to each
other. Such hybridization conditions are described in, for example
but not limited to, Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.; Basic Methods in
Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y.
(1986), pp. 75-78, and 84-87; and Molecular Cloning, Cold Spring
Harbor Laboratory, N.Y. (1982), pp. 387-389, and are well known to
those skilled in the art. A preferred, non-limiting example of
stringent hybridization conditions is hybridization in 6.times.
sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68.degree.
C. followed by one or more washes in 2.times.SSC, 0.5% SDS at room
temperature. Another preferred, non-limiting example of stringent
hybridization conditions is hybridization in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.2.times.SSC, 0.1%
SDS at about 50-65.degree. C.
[0025] The term "variant" as used herein refers either to a
naturally occurring genetic mutant of hSARS or a recombinantly
prepared variation of hSARS each of which contain one or more
mutations in its genome compared to the hSARS of CCTCC-V200303. The
term "variant" may also refers either to a naturally occurring
variation of a given peptide or a recombinantly prepared variation
of a given peptide or protein in which one or more amino acid
residues have been modified by amino acid substitution, addition,
or deletion.
4. BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 shows a partial DNA sequence (SEQ ID NO:1) and its
deduced amino acid sequence (SEQ ID NO:2) obtained from the SARS
virus that has 57% homology to the RNA-dependent RNA polymerase
protein of known Coronaviruses.
[0027] FIG. 2 shows an electron micrograph of the novel hSARS virus
that has similar morphological characteristics of
coronaviruses.
[0028] FIG. 3 shows an immunofluorescent staining for IgG
antibodies that are specifically bound to the FrHK-4 cells infected
with the novel human respiratory virus of Coronaviridae.
[0029] FIG. 4 shows an electron micrograph of ultra-centrifuged
deposit of hSARS virus that was grown in the cell culture and
negatively stained with 3% potassium phospho-tungstate at pH
7.0.
[0030] FIG. 5A shows a thin-section electron micrograph of lung
biopsy of a patient with SARS; and FIG. 5B shows a thin section
electron micrograph of hSARS-infected cells.
[0031] FIG. 6 shows the result of phylogenetic analysis for the
partial protein sequence (215 amino acids; SEQ ID NO:2) of the
hSARS virus (GenBank accession number AY268070). The phylogenetic
tree is constructed by the neighbor-jointing method. The
horizontal-line distance represents the number of sites at which
the two sequences compared are different. Bootstrap values are
deducted from 500 replicates.
[0032] FIG. 7A shows an amplification plot of fluorescence
intensity against the PCR cycle in a real-time quantitative PCR
assay that can detect a hSARS virus in samples quantitatively. The
copy numbers of input plasmid DNA in the reactions are indicated.
The X-axis denotes the cycle number of a quantitative PCR assay and
the Y-axis denotes the fluorescence intensity (FI) over the
backgroud. FIG. 7B shows the result of a melting curve analysis of
PCR products from clinical samples. Signals from positive (+ve)
samples, negative (-ve) samples and water control (water) are
indicated. The X-axis denotes the temperature (.degree. C.) and the
Y-axis denotes the fluorescence intensity (F1) over the
background.
[0033] FIG. 8 shows another partial DNA sequence (SEQ ID NO:11) and
its deduced amino acid sequence (SEQ ID NO:12) obtained from the
SARS virus.
[0034] FIG. 9 shows yet another partial DNA sequence (SEQ ID NO:13)
and its deduced amino acid sequence (SEQ ID NO:14) obtained from
the SARS virus.
[0035] FIG. 10 shows the entire genomic DNA sequence (SEQ ID NO:15)
of the SARS virus.
[0036] FIG. 11 shows the deduced amino acid sequences obtained from
SEQ ID NO:15 in three frames (see SEQ ID NOS:16, 240 and 737). An
asterisk (*) indicates a stop codon which marks the end of a
peptide. The first-frame amino acid sequences: SEQ ID NOS:17-239;
the second-frame amino acid sequences: SEQ ID NOS:241-736; and the
third-frame amino acid sequences: SEQ ID NO:738-1107.
[0037] FIG. 12 shows the deduced amino acid sequences obtained from
the complement of SEQ ID NO:15 in three frames (see SEQ ID
NOS:1108, 1590 and 1965). An asterisk (*) indicates a stop codon
which marks the end of a peptide. The first-frame amino acid
sequences: SEQ ID NOS:1109-1589; the second-frame amino acid
sequences: SEQ ID NOS:1591-1964; and the third-frame amino acid
sequences: SEQ ID NO:1966-2470.
[0038] FIG. 13 shows the N-gene primer sequences (which amplifies
nucleotides at position 29247-29410 of SEQ ID NO:2471). 150# (SEQ
ID NO:2475); 200# (SEQ ID NO:2476); and S-gene primer sequences
(which amplifies nucleotides at position 24751 to 25049 of SEQ ID
NO:2473). 131# (SEQ ID NO:2477); 132# (SEQ ID NO:2478).
[0039] FIG. 14A shows the nucleic acid sequence of the N-gene (SEQ
ID NO:2471). FIG. 14B shows the amino acid sequence of the N-gene
(SEQ ID NO:2472).
[0040] FIG. 15A shows the nucleic acid sequence of the S-gene (SEQ
ID NO:2473). FIG. 15B shows the amino acid sequence of the S-gene
(SEQ ID NO:2474).
[0041] FIG. 16 shows the genome organization and transcription
strategy of SARS-CoV HK-39. Genomic and mRNA transcripts are capped
(black circles), carry leader sequences (vertical lines)at
5'proximal and are polyadenylated (A.sup.15). Arrows point the
position of the intergenic sequence, 5'-CTAAACGAAC-3' (SEQ ID
NO:2479). After release of the positive-sense genomic RNA in the
cytoplasm of host cell, the viral RNA-dependent RNA polymerase,
encoded from ORF 1a and 1b, is synthesized. It carries out
transcription of a full-length complementary (negative-sense) RNA,
from which new genomic RNA, an overlapping set of subgenomic mRNA
transcripts, and leader RNA are synthesized. Note that all
transcripts are preceded with common 5' leader sequences and common
3' ends. ORF1a and 1b--RNA-dependent RNA polymerase; S--the major
peplomer glycoprotein; M--transmembrane glycoprotein;
N--nucleocapsid; X1, X2, X3--putative proteins.
[0042] FIG. 17 shows a construct map of pSARSCoV-ORF 1b-N. PCR
products amplified from ORF1b (1b) and N gene of SARS-CoV were
co-ligated into a cloning vector pCR2.1-TOPO (Invitrogen). The
nucleotide (nt) numbers corresponds to the positions in the
sequence of HK-39 strain SARS-CoV (AY278491). Shadowed areas
indicate the amplicons by the primers used in diagnostic test
(i.e., SEQ ID NOS:2480 and 2481).
[0043] FIG. 18 shows a photo of an agarose gel after
electrophoresis of total RNA extracted from SARS patients using SV
Total RNA isolation system. The extracted RNA was then subjected to
a reverse-transcription polymerase chain reaction (RT-PCR) assay
for the detection of coronavirus in the patients.
[0044] FIG. 19 shows the effect of potential inhibitors in Reverse
Transcription Polymerase Chain Reaction (RT-PCR). To remove
potential inhibitors, total RNA eluted from SV96 Binding Plate was
precipitated with 95% ethanol and 3 M sodium acetate and resuspend
in 12 .mu.l of nuclease-free water. RT-PCR was performed with
actin-F (SEQ ID NO:2482) and actin-R (SEQ ID NO:2483) primers.
Numbers indicated were the number of pig kidney epithelial (PK-15)
cell added in the sample as an internal control. There was no DNA
fragment amplified with untreated RNA samples.
[0045] FIG. 20 shows the primers used for amplifying various genes.
SRS251 (SEQ ID NO:2480) and SRS252 (SEQ ID NO:2481) amplified a 225
base pair fragment from the region of N-gene that showed no
homology to other coronavirus. coro3 (SEQ ID NO:3) and coro4 (SEQ
ID NO:4) amplified RNA-dependent RNA polymerase (1b gene) as a
control. Actin-F (SEQ ID NO:2482) and actin-R (SEQ ID NO:2483)
amplified a 745 base pair fragment from P-actin gene as internal
control for PCR assays.
[0046] FIG. 21A shows Amplification plot of fluorescence Intensity
against the number of PCR cycles. Black lines show the dynamic
range of N-gene specific PCR with serially diluted plasmid
construct from 10.sup.1 to 10.sup.6 copies. NPA samples from
non-SARS patients, including patients suffering from adenovirus
(n=5), respiratory syncytial virus (n=5), human metapneumovirus
(n=5), influenza A virus (n=5), or influenza B virus (n=5)
infection, are shown in gray lines. Lines with triangles denotes
the SARS-CoV positive NPA samples; NTC represents no template
control; X-axis indicates the cycle number of quantitative PCR
performed, while Y-axis represents the fluorescence intensity
(FAM-400) over background signal (Delta Rn). Inlet shows the
melting curve analysis of the PCR products. Signals from positive
(+ve), negative (-ve) samples and no template control are
indicated. X-axis indicates the temperature (.degree. C.), while
Y-axis represents the fluorescence intensity (Delta Rn). FIG. 21B
shows comparison of dynamic ranges of N-gene and 1b-gene specific
PCRs. Dynamic ranges of both N-gene and 1b-gene PCR were obtained
with same plasmid construct in which 1:1 ratio of corresponding
amplicons were subcloned. Serially diluted plasmid with copy number
ranged from 10.sup.-1 to 10.sup.5 copies was used as template in
both PCRs. Lines with triangles denotes N-gene specific PCR while
the gray lines indicates 1b-gene specific PCR. Inlet shows Ct
values.+-.standard deviation in triplicate set of experiment of
both PCRs with different copy numbers of template used. NTC
represents no template control; X-axis indicated the cycle number
of quantitative PCR performed, while Y-axis represents fluorescence
intensity.
[0047] FIGS. 22A and 22B show an amplification curve and a melting
curve, respectively, of real-time quantitative PCR specific to 1b
(using the primers having SEQ ID NOS:3 and 4) and N gene (using the
primers having SEQ ID NOS:2480 and 2481) of SARS CoV. FIG. 22A:
Amplification plot of fluorescence intensity against the number of
PCR cycles. One (1) .mu.l of cDNA from a NPA, tracheal dispersion
and lung biopsy of patients with clinical symptoms were used as
template in each PCR. Fifty (50) cycles of PCR were performed to
achieve the saturation phase of the reaction. X-axis indicates the
cycle number of quantitative PCR performed, while y-axis represents
the fluorescence intensity (FAM-490) over background signal.
Horizontal gray line indicates the calculated threshold value by
maximum curvature approach, and the baseline cycle Ct was
calculated automatically. Inlet shows half-maximal fluorescence
value ({fraction (1/2)} max) and Ct of both PCR with cDNA from
various tissue isolated from a key patient (patient A indicated in
New Engl. J. Med. 348:1967-76 (by Drosten et al., 2003) in three
different time points. NPA=nasopharyngeal aspirate; TW=tracheal
wash; LW=lung wash. FIG. 22B: Melting curves of PCR products.
Melting curve analysis was carried out after 10-minute
further-extension step of the reaction. The temperature was raised
from 56.degree. C. to 94.degree. C. by 76 increments of 0.5.degree.
C. each, while each set-point temperature had been held for 7
seconds for data collection and analysis. Melting temperature of
1b- and N-gene specific PCR products was 80.5.degree. C. and
85.5.degree. C. respectively. X-axis indicates the temperature in
degree Celsius while Y-axis represents the fluorescence intensity
(FAM-490) over background signal. One (1) .mu.l of water was used
as no template control in the reaction.
[0048] FIG. 23 shows the diagnostic result of 48 clinical samples
using the primers having SEQ ID NOS:2480 and 2481, respectively,
with .beta.-actin PCR as an internal control. Upper bands in each
row showed a 745 bp DNA fragment amplified with actin-F and
actin-R, while lower bands were the amplicons by primers specific
for N-gene of SARS coronavirus (225 bp). -ve control (water) and
+ve control (cDNA from SARS coronavirus infected vero cell) of the
assay were indicated. Five (5) .mu.l of PCR products of both
reactions were mixed and loaded into the sample well in a 2%
agarose gel. M=1 kb plus molecular marker (Invitrogen).
[0049] FIG. 24 shows Northern Blot analysis of SARS-CoV total RNA.
Total RNA of SARS-CoV was extracted from SARS-CoV infected Vero E6
cell. RNA was separated in a 1% denaturing gel containing 6.29%
formaldehyde. Afterwards RNA was transferred to positively charged
nylon membrane and hybridized with digoxigenin-labelled PCR
fragments specific to 1b, S, M and N genes, respectively. Lane
1--1b; lane 2--S; lane 3--M; lane 4--N. Vertical bar showed the
molecular size reference. Arrows indicates the transcripts
hybridized with N probe. Signals were analyzed by
chemiluminescence.
[0050] FIG. 25 shows the DNA probes used in Nothern blot analysis.
The probes for 1b gene (nt 18057-18222; SEQ ID NO:2484), S gene (nt
21920-22107; SEQ ID NO:2485), M gene (nt 25867-26996; SEQ ID
NO:2486), and N gene (nt 28658-28883; SEQ ID NO:2487) are
shown.
5. DETAILED DESCRIPTION OF THE INVENTION
[0051] The present inventors developed a rapid, high-throughput
reverse transcription-PCR diagnostic test for SARS associated
coronavirus (SARS-CoV). 3' region of the hSARS virus genome
including the Nucleocapsid gene (N-gene) represents a sensitive
molecular marker which can be used in addition to 1b gene to
increase the sensitivity of the test. An internal control using
PK-15 cells may be employed to ensure the integrity of RNA during
its extraction process and cDNA synthesis, thus eliminating false
negative results.
[0052] In mouse hepatitis virus (MHV), a typical member of the
genus Coronavirus, both genomic RNA and mRNA transcripts are capped
and with common 3' ends and common leader sequences on their 5'
ends. With this unique transcription strategy, the copy numbers of
different viral genes during proliferation of virus in its host are
different (FIG. 19). N gene that encodes for the nucleocapsid has
the most abundant copy number during virus replication as all
transcripts may carry nucleotide sequence from N gene, although
they are not all in-frame for translation for this gene product.
The present inventors have discovered a diagnostic assay that is
based on the 3' region, including the N-gene, of the viral genome
provide a more sensitive assay than the rest of the viral genome.
Accordingly, in preferred embodiments, nucleic acid molecules that
may be used for a diagnostic assay comprise nucleic acid sequence
of nucleotide position 18000 to 29742 of SEQ ID NO:15, or portions
thereof. The portions may comprise 15, 20, 25, 30, 35, 40, 45, 100,
150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 750,
800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 of nucleotides
having nucleic acid sequence from nucleotide position 18000 to
29742 of SEQ ID NO:15. In other preferred embodiments, nucleic acid
molecules that may be used for a diagnostic assay comprising
nucleic acid sequence of nucleotide position 28658 to 28883 or
29247-29410 of SEQ ID NO:15.
[0053] Nasopharyngeal aspirate (NPA) and stool samples were
obtained from SARS suspected patients with major clinical symptoms
and significant history of close contact with infected patients.
Total RNA was extracted from the subject samples, together with
PK-15 cell as an internal control. Samples were analyzed by the
reverse-transcription-PCR assay. Northern blot analysis was
performed to show different subgenomic transcripts of the virus.
Real-time quantitative PCR was employed to compare the sensitivity
of two loci used in this diagnostic assay. In specific embodiments,
PCR inhibitor was removed with ethanol precipitation after RNA
extraction process.
[0054] In preferred embodiments, the present invention provides a
method for detecting the presence or absence of nucleic acid of the
N-gene in a biological sample. The method involves obtaining a
biological sample from various sources and contacting the sample
with a compound or an agent capable of detecting a nucleic acid
(e.g., mRNA, genomic RNA) of the N-gene of the hSARS virus such
that the presence of the N-gene is detected in the sample. In
preferred embodiments, the N-gene may be detected using a labeled
nucleic acid probe comprising of the nucleotide sequence of SEQ ID
NO:2471, complement thereof, or a portion thereof. The portion may
be 10, 20, 30, 40, 50, 100, 200, 400, 500, 600, 800, 1000, 1200
nucleotides in length. In preferred embodiments, primers comprising
nucleotide sequence of SEQ ID NOS:2475 and/or 2476 or SEQ ID
NOS:2480 and/or 2481 may be used to amplify a portion of the N-gene
for detection.
[0055] A preferred agent for detecting hSARS mRNA or genomic RNA of
the invention is a labeled nucleic acid probe capable of
hybridizing to mRNA or genomic RNA encoding a polypeptide of the
invention. The nucleic acid probe can be, for example, a nucleic
acid molecule comprising or consisting of the nucleotide sequence
or SEQ ID NO:1, 11, 13, 15, 2471 or 2473, complement thereof, or a
portion thereof, such as an oligonucleotide of at least 15, 20, 25,
30, 50, 100, 250, 500, 750, 1,000 or more contiguous nucleotides in
length and sufficient to specifically hybridize under stringent
conditions to a hSARS mRNA or genomic RNA.
[0056] In another preferred specific embodiment, the presence of
N-gene is detected in the sample by an reverse transcription
polymerase chain reaction (RT-PCR) using the primers that are
constructed based on a partial nucleotide sequence of the N-gene or
a genomic nucleic acid sequence of SEQ ID NO:15, or based on a
nucleotide sequence of SEQ ID NO:1, 11, 13, 15, 2471, or 2473. In a
non-limiting specific embodiment, preferred primers to be used in a
RT-PCR method are: 5'-TACACACCTCAGC-GTTG-3' (SEQ ID NO:3) and/or
5'-CACGAACGTGACG-AAT-3' (SEQ ID NO:4), in the presence of 2.5 mM
MgCl.sub.2 and the thermal cycles are, for example, but not limited
to, 94.degree. C. for 8 min followed by 40 cycles of 94.degree. C.
for 1 min, 50.degree. C. for 1 min, 72.degree. C. for 1 min (also
see Sections 6.7 and 6.8 infra). In preferred embodiments, the
primers comprise nucleic acid sequence of SEQ ID NOS:2475 and 2476.
In another preferred embodiment, the primers comprise nucleic acid
sequence of SEQ ID NOS:2480 and 2481. In preferred embodiments, the
thermal cycles are 94.degree. C. for 10 min followed by 40 cycles
of 94.degree. C. for 30 seconds, 56.degree. C. for 30 seconds,
72.degree. C. for 30 seconds, 72.degree. C. for 10 minutes. In
another preferred embodiment, the thermal cycles are 94.degree. C.
for 3 min followed by 40 cycles of 94.degree. C. for 30 seconds,
56.degree. C. for 30 seconds, 72.degree. C. for 30 seconds,
72.degree. C. for 10 minutes. In more preferred specific
embodiment, the present invention provides a real-time quantitative
PCR assay to detect the presence of hSARS virus in a biological
sample by subjecting the cDNA obtained by reverse transcription of
the extracted total RNA from the sample to PCR reactions using the
specific primers, such as those having nucleotide sequences of SEQ
ID NOS:3 and 4, and a fluorescence dye, such as SYBR.RTM. Green I,
which fluoresces when bound non-specifically to double-stranded
DNA. The fluorescence signals from these reactions are captured at
the end of extension steps as PCR product is generated over a range
of the thermal cycles, thereby allowing the quantitative
determination of the viral load in the sample based on an
amplification plot (see Section 6.7, infra).
[0057] In the preferred embodiment, the present invention provides
a method for detecting the presence or absence of nucleic acid of
the S-gene in a biological sample. The method involves obtaining a
biological sample from various sources and contacting the sample
with a compound or an agent capable of detecting a nucleic acid
(e.g., MRNA, genomic RNA) of the S-gene of the hSARS virus such
that the presence of the S-gene is detected in the sample. A
preferred agent for detecting hSARS mRNA or genomic RNA of the
invention is a labeled nucleic acid probe capable of hybridizing to
mRNA or genomic RNA encoding a polypeptide of the invention. The
nucleic acid probe can be, for example, a nucleic acid molecule
comprising or consisting of the nucleotide sequence or SEQ ID NO:1,
11, 13, 15, 2471, or 2473, or a portion thereof, such as an
oligonucleotide of at least 15, 20, 25, 30, 50, 100, 250, 500, 750,
1,000 or more contiguous nucleotides in length and sufficient to
specifically hybridize under stringent conditions to a hSARS mRNA
or genomic RNA.
[0058] In another preferred specific embodiment, the presence of
S-gene is detected in the sample by an reverse transcription
polymerase chain reaction (RT-PCR) using the primers that are
constructed based on a partial nucleotide sequence of the S-gene
(SEQ ID NO:2473).
[0059] In vitro techniques for detection of mRNA include northern
hybridizations, in situ hybridizations, RT-PCR, and RNase
protection. In vitro techniques for detection of genomic RNA
include nothern hybridizations, RT-PCT, and RNase protection.
[0060] The polynucleotides encoding the N-gene may be amplified
before they are detected. The term "amplified" refers to the
process of making multiple copies of the nucleic acid from a single
polynucleotide molecule. The amplification of polynucleotides can
be carried out in vitro by biochemical processes known to those of
skill in the art. The amplification agent may be any compound or
system that will function to accomplish the synthesis of primer
extension products, including enzymes. Suitable enzymes for this
purpose include, for example, E. coli DNA polymerase I, Taq
polymerase, Klenow fragment of E. coli DNA polymerase I, T4 DNA
polymerase, other available DNA polymerases, polymerase muteins,
reverse transcriptase, ligase, and other enzymes, including
heat-stable enzymes (i.e., those enzymes that perform primer
extension after being subjected to temperatures sufficiently
elevated to cause denaturation). Suitable enzymes will facilitate
combination of the nucleotides in the proper manner to form the
primer extension products that are complementary to each mutant
nucleotide strand. Generally, the synthesis will be initiated at
the 3'-end of each primer and proceed in the 5'-direction along the
template strand, until synthesis terminates, producing molecules of
different lengths. There may be amplification agents, however, that
initiate synthesis at the 5'-end and proceed in the other
direction, using the same process as described above. In any event,
the method of the invention is not to be limited to the embodiments
of amplification described herein.
[0061] One method of in vitro amplification, which can be used
according to this invention, is the polymerase chain reaction (PCR)
described in U.S. Pat. Nos. 4,683,202 and 4,683,195. The term
"polymerase chain reaction" refers to a method for amplifying a DNA
base sequence using a heat-stable DNA polymerase and two
oligonucleotide primers, one complementary to the (+)-strand at one
end of the sequence to be amplified and the other complementary to
the (-)-strand at the other end. Because the newly synthesized DNA
strands can subsequently serve as additional templates for the same
primer sequences, successive rounds of primer annealing, strand
elongation, and dissociation produce rapid and highly specific
amplification of the desired sequence. The polymerase chain
reaction is used to detect the presence of polynucleotides encoding
cytokines in the sample. Many polymerase chain methods are known to
those of skill in the art and may be used in the method of the
invention. For example, DNA can be subjected to 30 to 35 cycles of
amplification in a thermocycler as follows: 95.degree. C. for 30
sec, 52.degree. to 60.degree. C. for 1 min, and 72.degree. C. for 1
min, with a final extension step of 72.degree. C. for 5 min. For
another example, DNA can be subjected to 35 polymerase chain
reaction cycles in a thermocycler at a denaturing temperature of
95.degree. C. for 30 sec, followed by varying annealing
temperatures ranging from 54-58.degree. C. for 1 min, an extension
step at 70.degree. C. for 1 min and a final extension step at
70.degree. C.
[0062] The primers for use in amplifying the N-gene or S-gene of
the invention may be prepared using any suitable method, such as
conventional phosphotriester and phosphodiester methods or
automated embodiments thereof so long as the primers are capable of
hybridizing to the polynucleotides of interest. One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066. The exact length of primer
will depend on many factors, including temperature, buffer, and
nucleotide composition. The primer must prime the synthesis of
extension products in the presence of the inducing agent for
amplification.
[0063] Primers used according to the method of the invention are
complementary to each strand of nucleotide sequence to be
amplified. The term "complementary" means that the primers must
hybridize with their respective strands under conditions, which
allow the agent for polymerization to function. In other words, the
primers that are complementary to the flanking sequences hybridize
with the flanking sequences and permit amplification of the
nucleotide sequence. Preferably, the 3' terminus of the primer that
is extended has perfectly base paired complementarity with the
complementary flanking strand. Primers and probes for
polynucleotides encoding N-gene or S-gene of the present invention
can be developed using known methods combined with the present
disclosure.
[0064] Those of ordinary skill in the art will know of various
amplification methodologies that can also be utilized to increase
the copy number of target nucleic acid. The polynucleotides
detected in the method of the invention can be further evaluated,
detected, cloned, sequenced, and the like, either in solution or
after binding to a solid support, by any method usually applied to
the detection of a specific nucleic acid sequence such as another
polymerase chain reaction, oligomer restriction (Saiki et al.,
Bio/Technology 3:1008-1012 (1985)), allele-specific oligonucleotide
(ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sci. USA 80:
278 (1983), oligonucleotide ligation assays (OLAs) (Landegren et
al., Science 241:1077 (1988)), RNAse Protection Assay and the like.
Molecular techniques for DNA analysis have been reviewed (Landegren
et al, Science 242: 229-237 (1988)). Following DNA amplification,
the reaction product may be detected by Southern blot analysis,
without using radioactive probes. In such a process, for example, a
small sample of DNA containing the polynucleotides obtained from
the tissue or subject are amplified, and analyzed via a Southern
blotting technique. The use of non-radioactive probes or labels is
facilitated by the high level of the amplified signal. In one
embodiment of the invention, one nucleoside triphosphate is
radioactively labeled, thereby allowing direct visualization of the
amplification product by autoradiography. In another embodiment,
amplification primers are fluorescently labeled and run through an
electrophoresis system. Visualization of amplified products is by
laser detection followed by computer assisted graphic display,
without a radioactive signal.
[0065] The methods of the present invention can involve a real-time
quantitative PCR assay, such as a Taqmang assay (Holland et al.,
Proc Natl Acad Sci USA, 88(16):7276 (1991); also see U.S. patent
application of Attorney Docket No. V9661.0078 filed Mar. 24, 2004,
which is incorporated by reference in its entirety). The assays can
be performed on an instrument designed to perform such assays, for
example those available from Applied Biosystems (Foster City,
Calif.). Primers and probes for such an assay can be designed
according to known procedures in the art.
[0066] The size of the primers used to amplify a portion of the
N-gene or S-gene is at least 10, 15, 20, 25, 30 nucleotide in
length. In particular, primers that amplify the N-gene or S-gene is
most preferred. Preferably, the GC ratio should be above 30, 35,
40, 45, 50, 55, 60% so as to prevent hair-pin structure on the
primer. Furthermore, the amplicon should be sufficiently long
enough to be detected by standard molecular biology
methodologies.
[0067] Preferably, the amplicon is at least 40, 60, 100, 200, 300,
400, 500, 600, 800, 1000 base pair in length.
[0068] In a specific embodiment, the methods further involve
obtaining a control sample from a control subject, contacting the
control sample with a compound or agent capable of detecting N-gene
or S-gene, such that the presence of mRNA or genomic RNA encoding
the N-gene or S-gene is detected in the sample, and comparing the
presence (or absence) of N-gene or S-gene, or mRNA or genomic RNA
encoding the polypeptide in the control sample with the presence of
N-gene or S-gene, or mRNA or genomic DNA encoding the polypeptide
in the test sample.
[0069] The invention also encompasses kits for detecting the
presence of N-gene nucleic acid in a test sample. The kit, for
example, can comprise a labeled compound or agent capable of
detecting a nucleic acid molecule encoding the polypeptide in a
test sample and, in certain embodiments, a means for determining
the amount of mRNA in the sample (an oligonucleotide probe which
binds to DNA or mRNA encoding the polypeptide).
[0070] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a polypeptide of the invention or to a sequence within the
N-gene; (2) a pair of primers useful for amplifying a nucleic acid
molecule containing the N-gene sequence. The kit can also comprise,
e.g., a buffering agent, a preservative, or a protein stabilizing
agent. The kit can also comprise components necessary for detecting
the detectable agent (e.g., an enzyme or a substrate). The kit can
also contain a control sample or a series of control samples which
can be assayed and compared to the test sample contained. Each
component of the kit is usually enclosed within an individual
container and all of the various containers are within a single
package along with instructions for use.
[0071] The present invention relates to the isolated N-gene and
S-gene of the hSARS virus. In a specific embodiment, the virus
comprises a nucleotide sequence of SEQ ID NO:1, 11, 13, 15, 2471,
and/or 2473. In a specific embodiment, the present invention
provides isolated nucleic acid molecules of the hSARS virus,
comprising, or, alternatively, consisting of the nucleotide
sequence of SEQ ID NO:1, 11, 13, 15, 2471, and/or, 2473, a
complement thereof or a portion thereof. In another specific
embodiment, the invention provides isolated nucleic acid molecules
which hybridize under stringent conditions, as defined herein, to a
nucleic acid molecule having the sequence of SEQ ID NO:1, 11, 13,
15, 2471, and/or 2473, or specific genes of known member of
Coronaviridae, or a complement thereof. In another specific
embodiment, the invention provides isolated polypeptides or
proteins that are encoded by a nucleic acid molecule comprising a
nucleotide sequence that is at least about 5, 10, 15, 20, 25, 30,
35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, or
more contiguous nucleotides of the nucleotide sequence of SEQ ID
NO:1, or a complement thereof. In another specific embodiment, the
invention provides isolated polypeptides or proteins that are
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,
200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, or more contiguous
nucleotides of the nucleotide sequence of SEQ ID NO:11, or a
complement thereof. In yet another specific embodiment, the
invention provides isolated polypeptides or proteins that are
encoded by a nucleic acid molecule comprising a nucleotide sequence
that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,
200, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more
contiguous nucleotides of the nucleotide sequence of SEQ ID NO:13,
or a complement thereof. In yet another specific embodiment, the
invention provides isolated polypeptides or proteins that are
encoded by a nucleic acid molecule comprising or, alternatively
consisting of a nucleotide sequence that is at least 5, 10, 15, 20,
25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,
1,200 or more contiguous nucleotides of the nucleotide sequence of
SEQ ID NO:2471, or a complement thereof. In yet another specific
embodiment, the invention provides isolated polypeptides or
proteins that are encoded by a nucleic acid molecule comprising or,
alternatively consisting of a nucleotide sequence that is at least
5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
1,050, 1,100, 1,150, 1,200, 2,000, 3,000, or more contiguous
nucleotides of the nucleotide sequence of SEQ ID NO:2473, or a
complement thereof. In yet another specific embodiment, the
invention provides isolated polypeptides or proteins that are
encoded by a nucleic acid molecule comprising or, alternatively
consisting of a nucleotide sequence that is at least 5, 10, 15, 20,
25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150,
1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000,
10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000,
18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000,
26,000, 27,000, 28,000, 29,000 or more contiguous nucleotides of
the nucleotide sequence of SEQ ID NO:15, or a complement thereof.
The polypeptides includes those shown in FIGS. 11 (SEQ ID
NOS:17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS: 1109-1589,
1591-1964 and 1966-2470) or having an amino acid sequence of SEQ ID
NO:2472 or 2474. The polypeptides or the proteins of the present
invention preferably have one or more biological activities of the
proteins encoded by the sequence of SEQ ID NO:1, 11, 13, 15, 2471
or 2473, or the polypeptides shown in FIGS. 11 and 12, or the
native viral proteins containing the amino acid sequences encoded
by the sequence of SEQ ID NO:1, 11, 13, 15, 2471 or 2473.
[0072] The present invention also relates to a method for
propagating the hSARS virus in host cells.
[0073] The invention further relates to the use of the sequence
information of the isolated virus for diagnostic and therapeutic
methods. In a specific embodiment, the invention provides the
entire nucleotide sequence of hSARS virus, CCTCC-V200303, SEQ ID
NO:15, or fragments, or complement thereof. Furthermore, the
present invention relates to a nucleic acid molecule that
hybridizes any portion of the genome of the hSARS virus,
CCTCC-V200303, or SEQ ID NO:15, under the stringent conditions. In
a specific embodiment, the invention provides nucleic acid
molecules which are suitable for use as primers consisting of or
comprising the nucleotide sequence of SEQ ID NO:1, 11, 13, 15, 2471
or 2473, or a complement thereof, or a portion thereof. In specific
embodiments, the primers comprise nucleotide sequence of SEQ ID NO:
2475, 2476, 2477, 2478, 2480 or 2481. In another specific
embodiment, the invention provides nucleic acid molecules which are
suitable for use as hybridization probes for the detection of
nucleic acids encoding a polypeptide of the invention, consisting
of or comprising the nucleotide sequence of SEQ ID NO:1, 11, 13,
15, 2471 or 2473, a complement thereof, or a portion thereof The
invention further relates to a kit comprising primers having
nucleic acid sequence of SEQ ID NOS:2475 and 2476; and SEQ ID
NOS:2480 and 2481, for the detection of N-gene. In another
embodiment, the invention relates to a kit comprising primers
having nucleic acid sequence of SEQ ID NOS:2477 and/or 2478 for the
detection of S-gene. In a preferred embodiment, the kit further
comprises reagents for the detection of genes not found in hSARS
virus as a negative control. The invention further encompasses
chimeric or recombinant viruses or viral proteins encoded by said
nucleotide sequences.
[0074] The invention further provides antibodies that specifically
bind a polypeptide of the invention encoded by the nucleotide
sequence of SEQ ID NO:1, 11, 13, 2471 or 2473, or a fragment
thereof, or any hSARS epitope. The invention further provides
antibodies that specifically bind a polypeptide having amino acid
sequence of SEQ ID NO: 2472 or 2474. The invention further provides
antibodies that specifically bind the polypeptides of the invention
encoded by the nucleotide sequence of SEQ ID NO:15, or the
polypeptides shown in FIGS. 11 and 12, or a fragment thereof, or
any hSARS epitope. Such antibodies include, but are not limited to
polyclonal, monoclonal, bi-specific, multi-specific, human,
humanized, chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, disulfide-linked Fvs,
intrabodies and fragments containing either a Vl or VH domain or
even a complementary determining region (CDR) that specifically
binds to a polypeptide of the invention.
[0075] In one embodiment, the invention provides methods for
detecting the presence, activity or expression of the hSARS virus
of the invention in a biological material, such as cells, blood,
saliva, urine, sputum, nasopharyngeal aspirates, and so forth. The
presence of the hSARS virus in a sample can be determined by
contacting the biological material with an agent which can detect
directly or indirectly the presence of the hSARS virus. In a
specific embodiment, the detection agents are the antibodies of the
present invention. In another embodiment, the detection agent is a
nucleic acid of the present invention.
[0076] In another embodiment, the invention provides vaccine
preparations comprising the hSARS virus, including recombinant and
chimeric forms of said virus, or subunits of the virus. In a
specific embodiment, the vaccine preparations comprise live but
attenuated hSARS virus with or without pharmaceutically acceptable
excipients, including adjuvants. In another specific embodiment,
the vaccine preparations comprise an inactivated or killed hSARS
virus with or without pharmaceutically acceptable excipients,
including adjuvants. The vaccine preparations of the present
invention may further comprise with adjuvants or Accordingly, the
present invention further provides methods of preparing recombinant
or chimeric forms of hSARS. In another specific invention, the
vaccine preparations of the present invention comprise one or more
nucleic acid molecules comprising or consisting of the sequence of
SEQ ID NO. 1, 11, 13, 15, 2471 and/or 2473, or a fragment thereof.
In another embodiment, the invention provides vaccine preparations
comprising one or more polypeptides of the invention encoded by a
nucleotide sequence comprising or consisting of the nucleotide
sequence of SEQ ID NO:1, 11, 13, 2471 and/or 2473, or the
polypeptides shown in FIGS. 11 and 12, or a fragment thereof. In
another embodiment, the invention provides vaccine preparations
comprising one or more polypeptides of the invention encoded by a
nucleotide sequence comprising or consisting of the nucleotide
sequence of SEQ ID NO:15, or a fragment thereof. Furthermore, the
present invention provides methods for treating, ameliorating,
managing, or preventing SARS by administering the vaccine
preparations or antibodies of the present invention alone or in
combination with antivirals [e.g., amantadine, rimantadine,
gancyclovir, acyclovir, ribavirin, penciclovir, oseltamivir,
foscarnet zidovudine (AZT), didanosine (ddI), lamivudine (3TC),
zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine,
indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, relenza,
tamiflu, pleconaril, interferons, etc.], steroids and
corticosteroids such as prednisone, cortisone, fluticasone and
glucocorticoid, antibiotics, analgesics, bronchodialaters, or other
treatments for respiratory and/or viral infections.
[0077] Furthermore, the present invention provides pharmaceutical
compositions comprising anti-viral agents of the present invention
and a pharmaceutically acceptable carrier. The present invention
also provides kits comprising pharmaceutical compositions of the
present invention.
[0078] In another aspect, the present invention provides methods
for screening anti-viral agents that inhibit the infectivity or
replication of hSARS virus or variants thereof.
[0079] 5.1 Recombinant and Chimeric hSARS Viruses
[0080] The present invention encompasses recombinant or chimeric
viruses encoded by viral vectors derived from the genome of hSARS
virus or natural variants thereof. In a specific embodiment, a
recombinant virus is one derived from the hSARS virus of deposit
accession no. CCTCC-V200303. In a specific embodiment, the virus
has a nucleotide sequence of SEQ ID NO:15. In another specific
embodiment, a recombinant virus is one derived from a natural
variant of hSARS virus. A natural variant of hSARS has a sequence
that is different from the genomic sequence (SEQ ID NO:15) of the
hSARS virus, CCTCC-V200303, due to one or more naturally occurred
mutations, including, but not limited to, point mutations,
rearrangements, insertions, deletions etc., to the genomic sequence
that may or may not result in a phenotypic change. In accordance
with the present invention, a viral vector which is derived from
the genome of the hSARS virus, CCTCC-V200303, is one that contains
a nucleic acid sequence that encodes at least a part of one ORF of
the hSARS virus. In a specific embodiment, the ORF comprises or
consists of a nucleotide sequence of SEQ ID NO:1, 11, 13, 2471,
2473, or a fragment thereof. In a specific embodiment, there are
more than one ORF within the nucleotide sequence of SEQ ID NO:15,
as shown in FIGS. 11 (SEQ ID NOS:16, 240 and 737) and 12 (SEQ ID
NOS:1108, 1590 and 1965), or a fragment thereof. In another
embodiment, the polypeptide encoded by the ORF comprises or
consists of an amino acid sequence of SEQ ID NO:2, 12, 14, 2472,
2474, or a fragment thereof, or shown in FIGS. 11 (SEQ ID NOS:
17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1 109-1589,
1591-1964 and 1966-2470), or a fragment thereof. In accordance with
the present invention these viral vectors may or may not include
nucleic acids that are non-native to the viral genome.
[0081] In another specific embodiment, a chimeric virus of the
invention is a recombinant hSARS virus which further comprises a
heterologous nucleotide sequence. In accordance with the invention,
a chimeric virus may be encoded by a nucleotide sequence in which
heterologous nucleotide sequences have been added to the genome or
in which endogenous or native nucleotide sequences have been
replaced with heterologous nucleotide sequences.
[0082] According to the present invention, the chimeric viruses are
encoded by the viral vectors of the invention which further
comprise a heterologous nucleotide sequence. In accordance with the
present invention a chimeric virus is encoded by a viral vector
that may or may not include nucleic acids that are non-native to
the viral genome. In accordance with the invention a chimeric virus
is encoded by a viral vector to which heterologous nucleotide
sequences have been added, inserted or substituted for native or
non-native sequences. In accordance with the present invention, the
chimeric virus may be encoded by nucleotide sequences derived from
different strains or variants of hSARS virus. In particular, the
chimeric virus is encoded by nucleotide sequences that encode
antigenic polypeptides derived from different strains or variants
of hSARS virus.
[0083] A chimeric virus may be of particular use for the generation
of recombinant vaccines protecting against two or more viruses (Tao
et al., J. Virol. 72, 2955-2961; Durbin et al., 2000, J. Virol. 74,
6821-6831; Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768
(1998); Teng et al., 2000, J. Virol. 74, 9317-9321). For example,
it can be envisaged that a virus vector derived from the hSARS
virus expressing one or more proteins of variants of hSARS virus,
or vice versa, will protect a subject vaccinated with such vector
against infections by both the native hSARS and the variant.
Attenuated and replication-defective viruses may be of use for
vaccination purposes with live vaccines as has been suggested for
other viruses. (See, PCT WO 02/057302, at pp. 6 and 23,
incorporated by reference herein).
[0084] In accordance with the present invention the heterologous
sequence to be incorporated into the viral vectors encoding the
recombinant or chimeric viruses of the invention include sequences
obtained or derived from different strains or variants of
hSARS.
[0085] In certain embodiments, the chimeric or recombinant viruses
of the invention are encoded by viral vectors derived from viral
genomes wherein one or more sequences, intergenic regions, termini
sequences, or portions or entire ORF have been substituted with a
heterologous or non-native sequence. In certain embodiments of the
invention, the chimeric viruses of the invention are encoded by
viral vectors derived from viral genomes wherein one or more
heterologous sequences have been inserted or added to the
vector.
[0086] The selection of the viral vector may depend on the species
of the subject that is to be treated or protected from a viral
infection. If the subject is human, then an attenuated hSARS virus
can be used to provide the antigenic sequences.
[0087] In accordance with the present invention, the viral vectors
can be engineered to provide antigenic sequences which confer
protection against infection by the hSARS and natural variants
thereof. The viral vectors may be engineered to provide one, two,
three or more antigenic sequences. In accordance with the present
invention the antigenic sequences may be derived from the same
virus, from different strains or variants of the same type of
virus, or from different viruses.
[0088] The expression products and/or recombinant or chimeric
virions obtained in accordance with the invention may
advantageously be utilized in vaccine formulations. The expression
products and chimeric virions of the present invention may be
engineered to create vaccines against a broad range of pathogens,
including viral and bacterial antigens, tumor antigens, allergen
antigens, and auto antigens involved in autoimmune disorders. In
particular, the chimeric virions of the present invention may be
engineered to create vaccines for the protection of a subject from
infections with hSARS virus and variants thereof.
[0089] In certain embodiments, the expression products and
recombinant or chimeric virions of the present invention may be
engineered to create vaccines against a broad range of pathogens,
including viral antigens, tumor antigens and autoantigens involved
in autoimmune disorders. One way to achieve this goal involves
modifying existing hSARS genes to contain foreign sequences in
their respective external domains. Where the heterologous sequences
are epitopes or antigens of pathogens, these chimeric viruses may
be used to induce a protective immune response against the disease
agent from which these determinants are derived.
[0090] Thus, the present invention relates to the use of viral
vectors and recombinant or chimeric viruses to formulate vaccines
against a broad range of viruses and/or antigens. The present
invention also encompasses recombinant viruses comprising a viral
vector derived from the hSARS or variants thereof which contains
sequences which result in a virus having a phenotype more suitable
for use in vaccine formulations, e.g., attenuated phenotype or
enhanced antigenicity. The mutations and modifications can be in
coding regions, in intergenic regions and in the leader and trailer
sequences of the virus.
[0091] The invention provides a host cell comprising a nucleic acid
or a vector according to the invention. Plasmid or viral vectors
containing the polymerase components of hSARS virus are generated
in prokaryotic cells for the expression of the components in
relevant cell types (bacteria, insect cells, eukaryotic cells).
Plasmid or viral vectors containing full-length or partial copies
of the hSARS genome will be generated in prokaryotic cells for the
expression of viral nucleic acids in-vitro or in-vivo. The latter
vectors may contain other viral sequences for the generation of
chimeric viruses or chimeric virus proteins, may lack parts of the
viral genome for the generation of replication defective virus, and
may contain mutations, deletions or insertions for the generation
of attenuated viruses. In addition, the present invention provides
a host cell infected with hSARS virus, for example, of deposit no.
CCTCC-V200303.
[0092] Infectious copies of hSARS (being wild type, attenuated,
replication-defective or chimeric) can be produced upon
co-expression of the polymerase components according to the
state-of-the-art technologies described above.
[0093] In addition, eukaryotic cells, transiently or stably
expressing one or more full-length or partial hSARS proteins can be
used. Such cells can be made by transfection (proteins or nucleic
acid vectors), infection (viral vectors) or transduction (viral
vectors) and may be useful for complementation of mentioned wild
type, attenuated, replication-defective or chimeric viruses.
[0094] The viral vectors and chimeric viruses of the present
invention may be used to modulate a subject's immune system by
stimulating a humoral immune response, a cellular immune response
or by stimulating tolerance to an antigen. As used herein, a
subject means: humans, primates, horses, cows, sheep, pigs, goats,
dogs, cats, avian species and rodents.
[0095] 5.2 Formulation of Vaccines and Antivirals
[0096] In a preferred embodiment, the invention provides a
proteinaceous molecule or hSARS virus specific viral protein or
functional fragment thereof encoded by a nucleic acid according to
the invention. Useful proteinaceous molecules are for example
derived from any of the genes or genomic fragments derivable from
the virus according to the invention, including envelop protein (E
protein), integral membrane protein (M protein), spike protein (S
protein), nucleocapsid protein (N protein), hemaglutinin esterase
(HE protein), and RNA-dependent RNA polymerase. Such molecules, or
antigenic fragments thereof, as provided herein, are for example
useful in diagnostic methods or kits and in pharmaceutical
compositions such as subunit vaccines. Particularly useful are
polypeptides encoded by the nucleotide sequence of SEQ ID NO:1, 11,
13, 15, 2471, 2473, or as shown in FIG. 11 (SEQ ID NOS:17-239,
241-736 and 738-1107) and FIG. 12 (SEQ ID NOS:1109-1589, 1591-1964
and 1966-2470), or having the amino acid sequence of SEQ ID NO:2472
or 2474, or antigenic fragments thereof for inclusion as antigen or
subunit immunogen, but inactivated whole virus can also be used.
Particularly useful are also those proteinaceous substances that
are encoded by recombinant nucleic acid fragments of the hSARS
genome, of course preferred are those that are within the preferred
bounds and metes of ORFs, in particular, for eliciting hSARS
specific antibody or T cell responses, whether in vivo (e.g. for
protective or therapeutic purposes or for providing diagnostic
antibodies) or in vitro (e.g. by phage display technology or
another technique useful for generating synthetic antibodies).
[0097] The invention provides vaccine formulations for the
prevention and treatment of infections with hSARS virus. In certain
embodiments, the vaccine of the invention comprises recombinant and
chimeric viruses of the hSARS virus. In certain embodiments, the
virus is attenuated.
[0098] In another embodiment of this aspect of the invention,
inactivated vaccine formulations may be prepared using conventional
techniques to "kill" the chimeric viruses. Inactivated vaccines are
"dead" in the sense that their infectivity has been destroyed.
Ideally, the infectivity of the virus is destroyed without
affecting its immunogenicity. In order to prepare inactivated
vaccines, the chimeric virus may be grown in cell culture or in the
allantois of the chick embryo, purified by zonal
ultracentrifugation, inactivated by formaldehyde or
.beta.-propiolactone, and pooled. The resulting vaccine is usually
inoculated intramuscularly.
[0099] Inactivated viruses may be formulated with a suitable
adjuvant in order to enhance the immunological response. Such
adjuvants may include but are not limited to mineral gels, e.g.,
aluminum hydroxide; surface active substances such as lysolecithin,
pluronic polyols, polyanions; peptides; oil emulsions; and
potentially useful human adjuvants such as BCG and Corynebacterium
parvum.
[0100] In another aspect, the present invention also provides DNA
vaccine formulations comprising a nucleic acid or fragment of the
hSARS virus, e.g., the virus having accession no. CCTCC-V200303, or
nucleic acid molecules having the sequence of SEQ ID NO:1, 11, 13,
15, 2471, 2473, or a fragment thereof. In another specific
embodiment, the DNA vaccine formulations of the present invention
comprises a nucleic acid or fragment thereof encoding the
antibodies which immunospecifically binds hSARS viruses. In DNA
vaccine formulations, a vaccine DNA comprises a viral vector, such
as that derived from the hSARS virus, bacterial plasmid, or other
expression vector, bearing an insert comprising a nucleic acid
molecule of the present invention operably linked to one or more
control elements, thereby allowing expression of the vaccinating
proteins encoded by said nucleic acid molecule in a vaccinated
subject. Such vectors can be prepared by recombinant DNA technology
as recombinant or chimeric viral vectors carrying a nucleic acid
molecule of the present invention (see also Section 5.1,
supra).
[0101] Various heterologous vectors are described for DNA
vaccinations against viral infections. For example, the vectors
described in the following references may be used to express hSARS
sequences instead of the sequences of the viruses or other
pathogens described; in particular, vectors described for hepatitis
B virus (Michel, M. L. et al., 1995, DAN-mediated immunization to
the hepatitis B surface antigen in mice: Aspects of the humoral
response mimic hepatitis B viral infection in humans, Proc. Natl.
Aca. Sci. USA 92:5307-5311; Davis, H. L. et al., 1993, DNA-based
immunization induces continuous seretion of hepatitis B surface
antigen and high levels of circulating antibody, Human Molec.
Genetics 2:1847-1851), HIV virus (Wang, B. et al., 1993, Gene
inoculation generates immune responses against human
imunodeficiency virus type 1, Proc. Natl. Acad. Sci. USA
90:4156-4160; Lu, S. et al., 1996, Simian immunodeficiency virus
DNA vaccine trial in macques, J. Virol. 70:3978-3991; Letvin, N. L.
et al., 1997, Potent, protective anti-HIV immune responses
generated by bimodal HIV envelope DNA plus protein vaccination,
Proc Natl Acad Sci USA. 94(17):9378-83), and influenza viruses
(Robinson, H l et al., 1993, Protection against a lethal influenza
virus challenge by immunization with a haemagglutinin-expressin- g
plasmid DNA, Vaccine 11:957-960; Ulmer, J. B. et al., Heterologous
protection against influenza by injection of DNA encoding a viral
protein, Science 259:1745-1749), as well as bacterial infections,
such as tuberculosis (Tascon, R. E. et al., 1996, Vaccination
against tuberculosis by DNA injection, Nature Med. 2:888-892;
Huygen, K. et al., 1996, Immunogenicity and protective efficacy of
a tuberculosis DNA vaccine, Nature Med., 2:893-898), and parasitic
infection, such as malaria (Sedegah, M., 1994, Protection against
malaria by immunization with plasmid DNA encoding circumsporozoite
protein, Proc. Natl. Acad. Sci. USA 91:9866-9870; Doolan, D. L. et
al., 1996, Circumventing genetic restriction of protection against
malaria with multigene DNA immunization: CD8+ T cell-interferon
.delta., and nitric oxide-dependent immunity, J. Exper. Med.,
1183:1739-1746).
[0102] Many methods may be used to introduce the vaccine
formulations described above. These include, but are not limited
to, oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and intranasal routes. Alternatively, it may be
preferable to introduce the chimeric virus vaccine formulation via
the natural route of infection of the pathogen for which the
vaccine is designed. The DNA vaccines of the present invention may
be administered in saline solutions by injections into muscle or
skin using a syringe and needle (Wolff J. A. et al., 1990, Direct
gene transfer into mouse muscle in vivo, Science 247:1465-1468;
Raz, E., 1994, Intradermal gene immunization: The possible role of
DNA uptake in the induction of cellular immunity to viruses, Proc.
Natl. Acd. Sci. USA 91:9519-9523). Another way to administer DNA
vaccines is called "gene gun" method, whereby microscopic gold
beads coated with the DNA molecules of interest is fired into the
cells (Tang, D. et al, 1992, Genetic immunization is a simple
method for eliciting an immune response, Nature 356:152-154). For
general reviews of the methods for DNA vaccines, see Robinson, H.
L., 1999, DNA vaccines: basic mechanism and immune responses
(Review), Int. J Mol. Med. 4(5):549-555; Barber, B., 1997,
Introduction: Emerging vaccine strategies, Seminars in Immunology
9(5):269-270; and Robinson, H. L. et al., 1997, DNA vaccines,
Seminars in Immunology 9(5):271-283.
[0103] 5.3 Attenuation of hSARS Virus or Variants Thereof
[0104] The hSARS virus or variants thereof of the invention can be
genetically engineered to exhibit an attenuated phenotype. In
particular, the viruses of the invention exhibit an attenuated
phenotype in a subject to which the virus is administered as a
vaccine. Attenuation can be achieved by any method known to a
skilled artisan. Without being bound by theory, the attenuated
phenotype of the viruses of the invention can be caused, e.g., by
using a virus that naturally does not replicate well in an intended
host species, for example, by reduced replication of the viral
genome, by reduced ability of the virus to infect a host cell, or
by reduced ability of the viral proteins to assemble to an
infectious viral particle relative to the wild type strain of the
virus.
[0105] The attenuated phenotypes of hSARS virus or variants thereof
can be tested by any method known to the artisan. A candidate virus
can, for example, be tested for its ability to infect a host or for
the rate of replication in a cell culture system. In certain
embodiments, growth curves at different temperatures are used to
test the attenuated phenotype of the virus. For example, an
attenuated virus is able to grow at 35.degree. C., but not at
39.degree. C. or 40.degree. C. In certain embodiments, different
cell lines can be used to evaluate the attenuated phenotype of the
virus. For example, an attenuated virus may only be able to grow in
monkey cell lines but not the human cell lines, or the achievable
virus titers in different cell lines are different for the
attenuated virus. In certain embodiments, viral replication in the
respiratory tract of a small animal model, including but not
limited to, hamsters, cotton rats, mice and guinea pigs, is used to
evaluate the attenuated phenotypes of the virus. In other
embodiments, the immune response induced by the virus, including
but not limited to, the antibody titers (e.g., assayed by plaque
reduction neutralization assay or ELISA) is used to evaluate the
attenuated phenotypes of the virus. In a specific embodiment, the
plaque reduction neutralization assay or ELISA is carried out at a
low dose. In certain embodiments, the ability of the hSARS virus to
elicit pathological symptoms in an animal model can be tested. A
reduced ability of the virus to elicit pathological symptoms in an
animal model system is indicative of its attenuated phenotype. In a
specific embodiment, the candidate viruses are tested in a monkey
model for nasal infection, indicated by mucous production.
[0106] The viruses of the invention can be attenuated such that one
or more of the functional characteristics of the virus are
impaired. In certain embodiments, attenuation is measured in
comparison to the wild type strain of the virus from which the
attenuated virus is derived. In other embodiments, attenuation is
determined by comparing the growth of an attenuated virus in
different host systems. Thus, for a non-limiting example, hSARS
virus or a variant thereof is said to be attenuated when grown in a
human host if the growth of the hSARS or variant thereof in the
human host is reduced compared to the non-attenuated hSARS or
variant thereof.
[0107] In certain embodiments, the attenuated virus of the
invention is capable of infecting a host, is capable of replicating
in a host such that infectious viral particles are produced. In
comparison to the wild type strain, however, the attenuated strain
grows to lower titers or grows more slowly. Any technique known to
the skilled artisan can be used to determine the growth curve of
the attenuated virus and compare it to the growth curve of the wild
type virus.
[0108] In certain embodiments, the attenuated virus of the
invention (e.g., a recombinant or chimeric hSARS) cannot replicate
in human cells as well as the wild type virus (e.g., wild type
hSARS) does. However, the attenuated virus can replicate well in a
cell line that lack interferon functions, such as Vero cells.
[0109] In other embodiments, the attenuated virus of the invention
is capable of infecting a host, of replicating in the host, and of
causing proteins of the virus of the invention to be inserted into
the cytoplasmic membrane, but the attenuated virus does not cause
the host to produce new infectious viral particles. In certain
embodiments, the attenuated virus infects the host, replicates in
the host, and causes viral proteins to be inserted in the
cytoplasmic membrane of the host with the same efficiency as the
wild type hSARS. In other embodiments, the ability of the
attenuated virus to cause viral proteins to be inserted into the
cytoplasmic membrane into the host cell is reduced compared to the
wild type virus. In certain embodiments, the ability of the
attenuated hSARS virus to replicate in the host is reduced compared
to the wild type virus. Any technique known to the skilled artisan
can be used to determine whether a virus is capable of infecting a
mammalian cell, of replicating within the host, and of causing
viral proteins to be inserted into the cytoplasmic membrane of the
host.
[0110] In certain embodiments, the attenuated virus of the
invention is capable of infecting a host. In contrast to the wild
type hSARS, however, the attenuated hSARS cannot be replicated in
the host. In a specific embodiment, the attenuated hSARS virus can
infect a host and can cause the host to insert viral proteins in
its cytoplasmic membranes, but the attenuated virus is incapable of
being replicated in the host. Any method known to the skilled
artisan can be used to test whether the attenuated hSARS has
infected the host and has caused the host to insert viral proteins
in its cytoplasmic membranes.
[0111] In certain embodiments, the ability of the attenuated virus
to infect a host is reduced compared to the ability of the wild
type virus to infect the same host. Any technique known to the
skilled artisan can be used to determine whether a virus is capable
of infecting a host.
[0112] In certain embodiments, mutations (e.g., missense mutations)
are introduced into the genome of the virus, for example, into the
sequence of SEQ ID NO:1, 11, 13, 15, 2471 or 2473, or to generate a
virus with an attenuated phenotype. Mutations (e.g., missense
mutations) can be introduced into the structural genes and/or
regulatory genes of the hSARS. Mutations can be additions,
substitutions, deletions, or combinations thereof. Such variant of
hSARS can be screened for a predicted functionality, such as
infectivity, replication ability, protein synthesis ability,
assembling ability, as well as cytopathic effect in cell cultures.
In a specific embodiment, the missense mutation is a cold-sensitive
mutation. In another embodiment, the missense mutation is a
heat-sensitive mutation. In another embodiment, the missense
mutation prevents a normal processing or cleavage of the viral
proteins.
[0113] In other embodiments, deletions are introduced into the
genome of the hSARS virus, which result in the attenuation of the
virus.
[0114] In certain embodiments, attenuation of the virus is achieved
by replacing a gene of the wild type virus with a gene of a virus
of a different species, of a different subgroup, or of a different
variant. In another aspect, attenuation of the virus is achieved by
replacing one or more specific domains of a protein of the wild
type virus with domains derived from the corresponding protein of a
virus of a different species. In certain other embodiments,
attenuation of the virus is achieved by deleting one or more
specific domains of a protein of the wild type virus.
[0115] When a live attenuated vaccine is used, its safety must also
be considered. The vaccine must not cause disease. Any techniques
known in the art that can make a vaccine safe may be used in the
present invention. In addition to attenuation techniques, other
techniques may be used. One non-limiting example is to use a
soluble heterologous gene that cannot be incorporated into the
virion membrane. For example, a single copy of the soluble version
of a viral transmembrane protein lacking the transmembrane and
cytosolic domains thereof, can be used.
[0116] Various assays can be used to test the safety of a vaccine.
For example, sucrose gradients and neutralization assays can be
used to test the safety. A sucrose gradient assay can be used to
determine whether a heterologous protein is inserted in a virion.
If the heterologous protein is inserted in the virion, the virion
should be tested for its ability to cause symptoms in an
appropriate animal model since the virus may have acquired new,
possibly pathological, properties.
[0117] 5.4 Adjuvants and Carrier Molecules
[0118] hSARS-associated antigens are administered with one or more
adjuvants. In one embodiment, the hSARS-associated antigen is
administered together with a mineral salt adjuvants or mineral salt
gel adjuvant. Such mineral salt and mineral salt gel adjuvants
include, but are not limited to, aluminum hydroxide (ALHYDROGEL,
REHYDRAGEL), aluminum phosphate gel, aluminum hydroxyphosphate
(ADJU-PHOS), and calcium phosphate.
[0119] In another embodiment, hSARS-associated antigen is
administered with an immunostimulatory adjuvant. Such class of
adjuvants, include, but are not limited to, cytokines (e.g.,
interleukin-2, interleukin-7, interleukin-12,
granulocyte-macrophage colony stimulating factor (GM-CSF),
interfereon-.gamma.-interleukin-1.beta. (IL-1.beta.), and
IL-1.beta. peptide or Sclavo Peptide), cytokine-containing
liposomes, triterpenoid glycosides or saponins (e.g., QuilA and
QS-21, also sold under the trademark STIMULON, ISCOPREP), Muramyl
Dipeptide (MDP) derivatives, such as
N-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold
under the trademark TERMURTIDE), GMDP,
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamnine,
N-acetylmuramyl-L-alanyl-D-
-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxy
phosphoryloxy)-ethylamine, muramyl tripeptide
phosphatidylethanolamine (MTP-PE), unmethylated CpG dinucleotides
and oligonucleotides, such as bacterial DNA and fragments thereof,
LPS, monophosphoryl Lipid A (3D-MLA sold under the trademark MPL),
and polyphosphazenes.
[0120] In another embodiment, the adjuvant used is a particular
adjuvant, including, but not limited to, emulsions, e.g., Freund's
Complete Adjuvant, Freund's Incomplete Adjuvant, squalene or
squalane oil-in-water adjuvant formulations, such as SAF and MF59,
e.g., prepared with block-copolymers, such as L-121
(polyoxypropylene/polyoxyetheylene) sold under the trademark
PLURONIC L-121, Liposomes, Virosomes, cochleates, and immune
stimulating complex, which is sold under the trademark ISCOM.
[0121] In another embodment, a microparticular adjuvant is used.,
Microparticulare adjuvants include, but are not limited to
biodegradable and biocompatible polyesters, homo- and copolymers of
lactic acid (PLA) and glycolic acid (PGA),
poly(lactide-co-glycolides) (PLGA) microparticles, polymers that
self-associate into particulates (poloxamer particles), soluble
polymers (polyphosphazenes), and virus-like particles (VLPs) such
as recombinant protein particulates, e.g., hepatitis B surface
antigen (HbsAg).
[0122] Yet another class of adjuvants that may be used include
mucosal adjuvants, including but not limited to heat-labile
enterotoxin from Escherichia coli (LT), cholera holotoxin (CT) and
cholera Toxin B Subunit (CTB) from Vibrio cholerae, mutant toxins
(e.g., LTK63 and LTR72), microparticles, and polymerized
liposomes.
[0123] In other embodiments, any of the above classes of adjuvants
may be used in combination with each other or with other adjuvants.
For example, non-limiting examples of combination adjuvant
preparations that can be used to administer the hSARS-associated
antigens of the invention include liposomes containing
immunostimulatory protein, cytokines, or T-cell and/or B-cell
peptides, or microbes with or without entrapped IL-2 or
microparticles containing enterotoxin. Other adjuvants known in the
art are also included within the scope of the invention (see
Vaccine Design: The Subunit and Adjuvant Approach, Chap. 7, Michael
F. Powell and Mark J. Newman (eds.), Plenum Press, New York, 1995,
which is incorporated herein in its entirety).
[0124] The effectiveness of an adjuvant may be determined by
measuring the induction of antibodies directed against an
immunogenic polypeptide containing a hSARS polypeptide epitope, the
antibodies resulting from administration of this polypeptide in
vaccines which are also comprised of the various adjuvants.
[0125] The polypeptides may be formulated into the vaccine as
neutral or salt forms. Pharmaceutically acceptable salts include
the acid additional salts (formed with free amino groups of the
peptide) and which are formed with inorganic acids, such as, for
example, hydrochloric or phosphoric acids, or organic acids such as
acetic, oxalic, tartaric, maleic, and the like. Salts formed with
free carboxyl groups may also be derived from inorganic bases, such
as, for example, sodium potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and the
like.
[0126] The vaccines of the invention may be multivalent or
univalent. Multivalent vaccines are made from recombinant viruses
that direct the expression of more than one antigen.
[0127] Many methods may be used to introduce the vaccine
formulations of the invention; these include but are not limited to
oral, intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal routes, and via scarification (scratching
through the top layers of skin, e.g., using a bifurcated
needle).
[0128] The patient to which the vaccine is administered is
preferably a mammal, most preferably a human, but can also be a
non-human animal including but not limited to cows, horses, sheep,
pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and
rats.
[0129] 5.5 Preparation of Antibodies
[0130] Antibodies which specifically recognize a polypeptide of the
invention, such as, but not limited to, polypeptides comprising the
sequence of SEQ ID NO:2, 12, 14, 2472, 2474, and polypeptides as
shown in FIGS. 11 (SEQ ID NOS:17-239, 241-736 and 738-1107) and 12
(SEQ ID NOS:1109-1589, 1591-1964 and 1966-2470), or hSARS epitope
or antigen-binding fragments thereof can be used for detecting,
screening, and isolating the polypeptide of the invention or
fragments thereof, or similar sequences that might encode similar
enzymes from the other organisms. For example, in one specific
embodiment, an antibody which immunospecifically binds hSARS
epitope, or a fragment thereof, can be used for various in vitro
detection assays, including enzyme-linked immunosorbent assays
(ELISA), radioimmunoassays, Western blot, etc., for the detection
of a polypeptide of the invention or, preferably, hSARS, in
samples, for example, a biological material, including cells, cell
culture media (e.g., bacterial cell culture media, mammalian cell
culture media, insect cell culture media, yeast cell culture media,
etc.), blood, plasma, serum, tissues, sputum, naseopharyngeal
aspirates, etc.
[0131] Antibodies specific for a polypeptide of the invention or
any epitope of hSARS may be generated by any suitable method known
in the art. Polyclonal antibodies to an antigen-of-interest, for
example, the hSARS virus from deposit no. CCTCC-V200303, or
comprises a nucleotide sequence of SEQ ID NO:15, can be produced by
various procedures well known in the art. For example, an antigen
can be administered to various host animals including, but not
limited to, rabbits, mice, rats, etc., to induce the production of
antisera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological
response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete) adjuvant, mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful adjuvants for humans such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are
also well known in the art.
[0132] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681
(Elsevier, N.Y., 1981) (both of which are incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0133] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with an antigen of
interest or a cell expressing such an antigen. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myelorna cells. Hybridomas are selected
and cloned by limiting dilution. The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding the antigen. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
inoculating mice intraperitoneally with positive hybridoma
clones.
[0134] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the complete light chain, and the variable
region, the CH1 region and the hinge region of the heavy chain.
[0135] The antibodies of the invention or fragments thereof can be
also produced by any method known in the art for the synthesis of
antibodies, in particular, by chemical synthesis or preferably, by
recombinant expression techniques.
[0136] The nucleotide sequence encoding an antibody may be obtained
from any information available to those skilled in the art (i.e.,
from Genbank, the literature, or by routine cloning and sequence
analysis). If a clone containing a nucleic acid encoding a
particular antibody or an epitope-binding fragment thereof is not
available, but the sequence of the antibody molecule or
epitope-binding fragment thereof is known, a nucleic acid encoding
the immunoglobulin may be chemically synthesized or obtained from a
suitable source (e.g., an antibody cDNA library, or a cDNA library
generated from, or nucleic acid, preferably poly A+ RNA, isolated
from any tissue or cells expressing the antibody, such as hybridoma
cells selected to express an antibody) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0137] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., supra; and Ausubel et al., eds., 1998, Current
Protocols in Molecular Biology, John Wiley & Sons, NY, which
are both incorporated by reference herein in their entireties), to
generate antibodies having a different amino acid sequence by, for
example, introducing amino acid substitutions, deletions, and/or
insertions into the epitope-binding domain regions of the
antibodies or any portion of antibodies which may enhance or reduce
biological activities of the antibodies.
[0138] Recombinant expression of an antibody requires construction
of an expression vector containing a nucleotide sequence that
encodes the antibody. Once a nucleotide sequence encoding an
antibody molecule or a heavy or light chain of an antibody, or
portion thereof has been obtained, the vector for the production of
the antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art as discussed in the previous
sections. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing antibody
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. The nucleotide sequence encoding the
heavy-chain variable region, light-chain variable region, both the
heavy-chain and light-chain variable regions, an epitope-binding
fragment of the heavy- and/or light-chain variable region, or one
or more complementarity determining regions (CDRs) of an antibody
may be cloned into such a vector for expression. Thus-prepared
expression vector can be then introduced into appropriate host
cells for the expression of the antibody. Accordingly, the
invention includes host cells containing a polynucleotide encoding
an antibody specific for the polypeptides of the invention or
fragments thereof.
[0139] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides or different selectable markers to ensure
maintenance of both plasmids. Alternatively, a single vector may be
used which encodes, and is capable of expressing, both heavy and
light chain polypeptides. In such situations, the light chain
should be placed before the heavy chain to avoid an excess of toxic
free heavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler,
Proc. Natl. Acad. Sci. USA, 77:2 197, 1980). The coding sequences
for the heavy and light chains may comprise cDNA or genomic
DNA.
[0140] In another embodiment, antibodies can also be generated
using various phage display methods known in the art. In phage
display methods, functional antibody domains are displayed on the
surface of phage particles which carry the polynucleotide sequences
encoding them. In a particular embodiment, such phage can be
utilized to display antigen binding domains, such as Fab and Fv or
disulfide-bond stabilized Fv, expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage,
including fd and M13. The antigen binding domains are expressed as
a recombinantly fused protein to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make the immunoglobulins, or fragments thereof, of the present
invention include those disclosed in Brinkman et al., J. Immunol.
Methods, 182:41-50, 1995; Ames et al., J. Immunol. Methods,
184:177-186, 1995; Kettleborough et al., Eur. J. Immunol.,
24:952-958, 1994; Persic et al., Gene, 187:9-18, 1997; Burton et
al., Advances in Immunology, 57:191-280, 1994; PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0141] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired fragments, and expressed in any desired host,
including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as described in detail below. For example,
techniques to recombinantly produce Fab, Fab' and F(ab).sub.2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques, 12(6):864-869, 1992; and Sawai et al., AJRI,
34:26-34, 1995; and Better et al., Science, 240:1041-1043, 1988
(each of which is incorporated by reference in its entirety).
Examples of techniques which can be used to produce single-chain
Fvs and antibodies include those described in U.S. Pat. Nos.
4,946,778 and 5,258,498; Huston et al., Methods in Enzymology,
203:46-88, 1991; Shu et al., PNAS, 90:7995-7999, 1993; and Skerra
et al., Science, 240:1038-1040, 1988.
[0142] Once an antibody molecule of the invention has been produced
by any methods described above, it may then be purified by any
method known in the art for purification of an immunoglobulin
molecule, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after
Protein A or Protein G purification, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard techniques for the purification of proteins.
Further, the antibodies of the present invention or fragments
thereof may be fused to heterologous polypeptide sequences
described herein or otherwise known in the art to facilitate
purification.
[0143] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a constant
region derived from a human immunoglobulin. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison,
Science, 229:1202, 1985; Oi et al., BioTechniques, 4:214 1986;
Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Pat.
Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated
herein by reference in their entireties. Humanized antibodies are
antibody molecules from non-human species that bind the desired
antigen having one or more complementarity determining regions
(CDRs) from the non-human species and framework regions from a
human immunoglobulin molecule. Often, framework residues in the
human framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature, 332:323, 1988, which are incorporated
herein by reference in their entireties. Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology,
28(4/5):489-498, 1991; Studnicka et al., Protein Engineering,
7(6):805-814, 1994; Roguska et al., Proc Natl. Acad. Sci. USA,
91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332),
all of which are hereby incorporated by reference in their
entireties.
[0144] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887
and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO
98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741,
each of which is incorporated herein by reference in its
entirety.
[0145] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar, Int. Rev. Immunol., 13:65-93, 1995. For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publications WO 98/24893;
WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598
877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and
5,939,598, which are incorporated by reference herein in their
entireties. In addition, companies such as Abgenix, Inc. (Fremont,
Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0146] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology, 12:899-903, 1988).
[0147] Antibodies fused or conjugated to heterologous polypeptides
may be used in in vitro immunoassays and in purification methods
(e.g., affinity chromatography) well known in the art. See e.g.,
PCT publication Number WO 93/21232; EP 439,095; Naramura et al.,
Immunol. Lett., 39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et
al., PNAS, 89:1428-1432, 1992; and Fell et al., J. Immunol.,
146:2446-2452, 1991, which are incorporated herein by reference in
their entireties.
[0148] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the
polypeptides of the invention or fragments, derivatives, analogs,
or variants thereof, or similar molecules having the similar
enzymatic activities as the polypeptide of the invention. Such
solid supports include, but are not limited to, glass, cellulose,
polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene.
[0149] 5.6 Pharmaceutical Compositions and Kits
[0150] The present invention encompasses pharmaceutical
compositions comprising anti-viral agents of the present invention.
In a specific embodiment, the anti-viral agent is an antibody which
immunospecifically binds and neutralize the hSARS virus or variants
thereof, or any proteins derived therefrom. In another specific
embodiment, the anti-viral agent is a polypeptide or nucleic acid
molecule of the invention. The pharmaceutical compositions have
utility as an anti-viral prophylactic agent and may be administered
to a subject where the subject has been exposed or is expected to
be exposed to a virus.
[0151] Various delivery systems are known and can be used to
administer the pharmaceutical composition of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the mutant viruses,
receptor mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.
Chem. 262:4429 4432). Methods of introduction include but are not
limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In a preferred embodiment, it may be desirable to introduce
the pharmaceutical compositions of the invention into the lungs by
any suitable route. Pulmonary administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with an
aerosolizing agent.
[0152] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) infected tissues.
[0153] In another embodiment, the pharmaceutical composition can be
delivered in a vesicle, in particular a liposome (see Langer, 1990,
Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.),
Liss, N.Y., pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.).
[0154] In yet another embodiment, the pharmaceutical composition
can be delivered in a controlled release system. In one embodiment,
a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201; Buchwald et al.,1980, Surgery 88:507; and
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
N.Y. (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol.
Chem. 23:61 (1983); see also Levy et al., 1985, Science 228:190;
During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.
Neurosurg. 71:105). In yet another embodiment, a controlled release
system can be placed in proximity of the composition's target,
i.e., the lung, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0155] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0156] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of an live attenuated,
inactivated or killed hSARS virus, or recombinant or chimeric hSARS
virus, and a pharmaceutically acceptable carrier. In a specific
embodiment, the term "pharmaceutically acceptable" means approved
by a regulatory agency of the Federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly in humans.
The term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which the pharmaceutical composition is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained release
formulations and the like. The composition can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. The
formulation should suit the mode of administration.
[0157] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0158] The pharmaceutical compositions of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with free carboxyl groups such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2 ethylamino ethanol,
histidine, procaine, etc.
[0159] The amount of the pharmaceutical composition of the
invention which will be effective in the treatment of a particular
disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20 500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose response curves derived from in vitro or animal model
test systems.
[0160] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0161] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In a preferred embodiment, the kit
contains an anti-viral agent of the invention, e.g., an antibody
specific for the polypeptides encoded by a nucleotide sequence of
SEQ ID NO:1, 11, 13, 15, 2471 or 2473, or as shown in FIGS. 11 (SEQ
ID NOS: 17-239, 241-736 and 738-1107) and 12 (SEQ ID NOS:1109-1589,
1591-1964 and 1966-2470), or any hSARS epitope, or a polypeptide or
protein of the present invention, or a nucleic acid molecule of the
invention, alone or in combination with adjuvants, antivirals,
antibiotics, analgesic, bronchodialaters, or other pharmaceutically
acceptable excipients.
[0162] The present invention further encompasses kits comprising a
container containing a pharmaceutical composition of the present
invention and instructions to for use.
[0163] 5.7 Detection Assays
[0164] The present invention provides a method for detecting an
antibody, which immunospecifically binds to the hSARS virus, in a
biological sample, for example blood, serum, plasma, saliva, urine,
etc., from a patient suffering from SARS. In a specific embodiment,
the method comprising contacting the sample with the hSARS virus,
for example, of deposit no. CCTCC-V200303, or having a genomic
nucleic acid sequence of SEQ ID NO:15, directly immobilized on a
substrate and detecting the virus-bound antibody directly or
indirectly by a labeled heterologous anti-isotype antibody. In
another specific embodiment, the sample is contacted with a host
cell which is infected by the hSARS virus, for example, of deposit
no. CCTCC-V200303, or having a genomic nucleic acid sequence of SEQ
ID NO:15, and the bound antibody can be detected by
immunofluorescent assay as described in Section 6.5, infra.
[0165] An exemplary method for detecting the presence or absence of
a polypeptide or nucleic acid of the invention in a biological
sample involves obtaining a biological sample from various sources
and contacting the sample with a compound or an agent capable of
detecting an epitope or nucleic acid (e.g., mRNA, genomic RNA) of
the hSARS virus such that the presence of the hSARS virus is
detected in the sample. A preferred agent for detecting hSARS mRNA
or genomic RNA of the invention is a labeled nucleic acid probe
capable of hybridizing to mRNA or genomic RNA encoding a
polypeptide of the invention. The nucleic acid probe can be, for
example, a nucleic acid molecule comprising or consisting of the
nucleotide sequence or SEQ ID NO:1, 11, 13, 15, 2471, or 2473, or a
portion thereof, such as an oligonucleotide of at least 15, 20, 25,
30, 50, 100, 250, 500, 750, 1,000 or more contiguous nucleotides in
length and sufficient to specifically hybridize under stringent
conditions to a hSARS mRNA or genomic RNA.
[0166] In another preferred specific embodiment, the presence of
hSARS virus is detected in the sample by an reverse transcription
polymerase chain reaction (RT-PCR) using the primers that are
constructed based on a partial nucleotide sequence of the genome of
hSARS virus, for example, that of deposit accession no.
CCTCC-V200303, or having a genomic nucleic acid sequence of SEQ ID
NO:15, or based on a nucleotide sequence of SEQ ID NO:1, 11, 13,
2471 or 2473. In a non-limiting specific embodiment, preferred
primers to be used in a RT-PCR method are: 5'-TACACACCTCAGC-GTTG-3'
(SEQ ID NO:3) and 5'-CACGAACGTGACG-AAT-3' (SEQ ID NO:4), in the
presence of 2.5 mM MgCl.sub.2 and the thermal cycles are, for
example, but not limited to, 94.degree. C. for 8 min followed by 40
cycles of 94.degree. C. for 1 min, 50.degree. C. for 1 min,
72.degree. C. for 1 min (also see Sections 6.7 and 6.8 infra). In
preferred embodiments, the primers comprise nucleic acid sequence
of SEQ ID NOS:2475 and 2476, or SEQ ID NOS:2480 and 2481. In
preferred embodiments, the thermal cycles are 94.degree. C. for 10
min followed by 40 cycles of 94.degree. C. for 30 seconds,
56.degree. C. for 30 seconds, 72.degree. C. for 30 seconds,
72.degree. C. for 10 minutes. In preferred embodiments, the primers
comprise nucleic acid sequence of SEQ ID NOS:2477 and 2478. In more
preferred specific embodiment, the present invention provides a
real-time quantitative PCR assay to detect the presence of hSARS
virus in a biological sample by subjecting the cDNA obtained by
reverse transcription of the extracted total RNA from the sample to
PCR reactions using the specific primers, such as those having
nucleotide sequences of SEQ ID NOS:3 and 4, and a fluorescence dye,
such as SYBR.RTM. Green I, which fluoresces when bound
non-specifically to double-stranded DNA. The fluorescence signals
from these reactions are captured at the end of extension steps as
PCR product is generated over a range of the thermal cycles,
thereby allowing the quantitative determination of the viral load
in the sample based on an amplification plot (see Section 6.7,
infra).
[0167] A preferred agent for detecting hSARS is an antibody that
specifically binds a polypeptide of the invention or any hSARS
epitope, preferably an antibody with a detectable label. Antibodies
can be polyclonal, or more preferably, monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be
used.
[0168] The term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The detection
method of the invention can be used to detect mRNA, protein (or any
epitope), or genomic RNA in a sample in vitro as well as in vivo.
For example, in vitro techniques for detection of mRNA include
northern hybridizations, in situ hybridizations, RT-PCR, and RNase
protection. In vitro techniques for detection of an epitope of
hSARS include enzyme linked immunosorbent assays (ELISAs), Western
blots, immunoprecipitations and immunofluorescence. In vitro
techniques for detection of genomic RNA include nothern
hybridizations, RT-PCT, and RNase protection. Furthermore, in vivo
techniques for detection of hSARS include introducing into a
subject organism a labeled antibody directed against the
polypeptide. For example, the antibody can be labeled with a
radioactive marker whose presence and location in the subject
organism can be detected by standard imaging techniques, including
autoradiography.
[0169] In a specific embodiment, the methods further involve
obtaining a control sample from a control subject, contacting the
control sample with a compound or agent capable of detecting hSARS,
e.g., a polypeptide of the invention or mRNA or genomic RNA
encoding a polypeptide of the invention, such that the presence of
hSARS or the polypeptide or mRNA or genomic RNA encoding the
polypeptide is detected in the sample, and comparing the presence
of hSARS or the polypeptide or mRNA or genomic RNA encoding the
polypeptide in the control sample with the presence of hSARS, or
the polypeptide or mRNA or genomic DNA encoding the polypeptide in
the test sample.
[0170] The invention also encompasses kits for detecting the
presence of hSARS or a polypeptide or nucleic acid of the invention
in a test sample. The kit, for example, can comprise a labeled
compound or agent capable of detecting hSARS or the polypeptide or
a nucleic acid molecule encoding the polypeptide in a test sample
and, in certain embodiments, a means for determining the amount of
the polypeptide or mRNA in the sample (e.g., an antibody which
binds the polypeptide or an oligonucleotide probe which binds to
DNA or mRNA encoding the polypeptide). Kits can also include
instructions for use.
[0171] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide of the invention or hSARS epitope; and,
optionally, (2) a second, different antibody which binds to either
the polypeptide or the first antibody and is conjugated to a
detectable agent.
[0172] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a polypeptide of the invention or to a sequence within the
hSARS genome or (2) a pair of primers useful for amplifying a
nucleic acid molecule containing an hSARS sequence. The kit can
also comprise, e.g., a buffering agent, a preservative, or a
protein stabilizing agent. The kit can also comprise components
necessary for detecting the detectable agent (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample contained. Each component of the kit is usually enclosed
within an individual container and all of the various containers
are within a single package along with instructions for use.
[0173] 5.8 Screening Assays to Identify Anti-Viral Agents
[0174] The invention provides methods for the identification of a
compound that inhibits the ability of hSARS virus to infect a host
or a host cell. In certain embodiments, the invention provides
methods for the identification of a compound that reduces the
ability of hSARS virus to replicate in a host or a host cell. Any
technique well-known to the skilled artisan can be used to screen
for a compound that would abolish or reduce the ability of hSARS
virus to infect a host and/or to replicate in a host or a host
cell.
[0175] In certain embodiments, the invention provides methods for
the identification of a compound that inhibits the ability of hSARS
virus to replicate in a mammal or a mammalian cell. More
specifically, the invention provides methods for the identification
of a compound that inhibits the ability of hSARS virus to infect a
mammal or a mammalian cell. In certain embodiments, the invention
provides methods for the identification of a compound that inhibits
the ability of hSARS virus to replicate in a mammalian cell. In a
specific embodiment, the mammalian cell is a human cell.
[0176] In another embodiment, a cell is contacted with a test
compound and infected with the hSARS virus. In certain embodiments,
a control culture is infected with the hSARS virus in the absence
of a test compound. The cell can be contacted with a test compound
before, concurrently with, or subsequent to the infection with the
hSARS virus. In a specific embodiment, the cell is a mammalian
cell. In an even more specific embodiment, the cell is a human
cell. In certain embodiments, the cell is incubated with the test
compound for at least 1 minute, at least 5 minutes at least 15
minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at
least 5 hours, at least 12 hours, or at least 1 day. The titer of
the virus can be measured at any time during the assay. In certain
embodiments, a time course of viral growth in the culture is
determined. If the viral growth is inhibited or reduced in the
presence of the test compound, the test compound is identified as
being effective in inhibiting or reducing the growth or infection
of the hSARS virus. In a specific embodiment, the compound that
inhibits or reduces the growth of the hSARS virus is tested for its
ability to inhibit or reduce the growth rate of other viruses to
test its specificity for the hSARS virus.
[0177] In one embodiment, a test compound is administered to a
model animal and the model animal is infected with the hSARS virus.
In certain embodiments, a control model animal is infected with the
hSARS virus without the administration of a test compound. The test
compound can be administered before, concurrently with, or
subsequent to the infection with the hSARS virus. In a specific
embodiment, the model animal is a mammal. In an even more specific
embodiment, the model animal can be, but is not limited to, a
cotton rat, a mouse, or a monkey. The titer of the virus in the
model animal can be measured at any time during the assay. In
certain embodiments, a time course of viral growth in the culture
is determined. If the viral growth is inhibited or reduced in the
presence of the test compound, the test compound is identified as
being effective in inhibiting or reducing the growth or infection
of the hSARS virus. In a specific embodiment, the compound that
inhibits or reduces the growth of the hSARS in the model animal is
tested for its ability to inhibit or reduce the growth rate of
other viruses to test its specificity for the hSARS virus.
6. EXAMPLES
[0178] The following examples illustrate the isolation and
identification of the novel hSARS virus. These examples should not
be construed as limiting.
[0179] Methods and Results
[0180] As a general reference, Wiedbrauk D l & Johnston S L G.
(Manual of Clinical Virology, Raven Press, New York, 1993) was
used.
[0181] 6.1 Clinical Subjects
[0182] The study included all 50 patients who fitted a modified
World Health Organization (WHO) definition of SARS and were
admitted to 2 acute regional hospitals in Hong Kong Special
Administrative Region (HKSAR) between Feb. 26 to Mar. 26, 2003
(WHO. Severe acute respiratory syndrome (SARS) Weekly Epidemiol
Rec. 2003; 78: 81-83). A lung biopsy from an additional patient,
who had typical SARS and was admitted to a third hospital, was also
included in the study. Briefly, the case definition for SARS was:
(i) fever of 38.degree. C. or more; (ii) cough or shortness of
breath; (iii) new pulmonary infiltrates on chest radiograph; and
(iv) either a history of exposure to a patient with SARS or absence
of response to empirical antimicrobial coverage for typical and
atypical pneumonia (beta-lactams and macrolides, fluoroquinolones
or tetracyclines).
[0183] Nasopharyngeal aspirates and serum samples were collected
from all patients. Paired acute and convalescent sera and feces
were available from some patients. Lung biopsy tissue from one
patient was processed for a viral culture, RT-PCR, routine
histopathological examination, and electron microscopy.
Nasopharyngeal aspirates, feces and sera submitted for
microbiological investigation of other diseases were included in
the study under blinding and served as controls.
[0184] The medical records were reviewed retrospectively by the
attending physicians and clinical microbiologists. Routine
hematological, biochemical and microbiological examinations,
including bacterial culture of blood and sputum, serological study
and collection of nasopharyngeal aspirates for virological tests,
were carried out.
[0185] 6.2 Cell Line
[0186] FRhK-4 (fetal rhesus monkey kidney) cells were maintained in
minimal essential medium (MEM) with 1% fetal calf serum, 1%
streptomycin and penicillin, 0.2% nystatin and 0.05% garamycin.
[0187] 6.3 Viral Infection
[0188] Two-hundred .mu.l of clinical (nasopharyngeal aspirates)
samples, from two patients (see the Result section, infra), in
virus transport medium were used to infect FRhk-4 cells. The
inoculated cells were incubated at 37.degree. C. for 1 hour. One ml
of MEM containing 1 .mu.g trypsin was then added to the culture and
the infected cells were incubated in a 37.degree. C. incubator
supplied with 5% carbon dioxide. Cytopathic effects were observed
in the infected cells after 2 to 4 days of incubation. The infected
cells were passaged into new FRhK-4 cells and cytopathic effects
were observed within 1 day after the inoculation. The infected
cells were tested by an immunofluorescent assay for influenza A.,
influenza B, respiratory syncytial virus, parainfluenza types 1, 2
and 3, adenovirus and human metapneumovirus (hMPV) and negative
results were obtained for all cases. The infected cells were also
tested by RT-PCR for influenza A and human metapneumovirus with
negative results.
[0189] 6.4 Virus Morphology
[0190] The infected cells prepared as described above were
harvested, pelleted by centrifugation and the cell pellets were
processed for thin-section transmitted electron microscopic
visualization. Viral particles were identified in the cells
infected with both clinical specimens, but not in control cells
which were not infected with the virus. Virions isolated from the
infected cells were about 70-100 nanometers (FIG. 2). Viral capsids
were found predominantly within the vesicles of the golgi and
endoplasmic reticulum and were not free in the cytoplasm. Virus
particles were also found at the cell membrane.
[0191] One virus isolate was ultracentrifuged and the cell pellet
was negatively stained using phosphotugstic acid. Virus particles
characteristic of Coronaviridae were thus visualized. Since the
human Coronaviruses hitherto recognized are not known to cause a
similar disease, the present inventors postulated that the virus
isolates represent a novel virus that infects humans.
[0192] 6.5 Antibody Response to the Isolated Virus
[0193] To further confirm that this novel virus is responsible for
causing SARS in the infected patients, blood serum samples from the
patients who were suffering from SARS were obtained and a
neutralization test was performed. Typically diluted serum
(.times.50, .times.200, .times.800 and .times.1600) was incubated
with acetone-fixed FRhK-4 cells infected with hSARS at 37.degree.
C. for 45 minutes. The incubated cells were then washed with
phosphate-buffered saline and stained with anti-human IgG-FITC
conjugated antibody. The cells were then washed and examined under
a fluorescent microscope. In these experiments, positive signals
were found in 8 patients who had SARS (FIG. 3), indicating that
these patients had an IgG antibody response to this novel human
respiratory virus of Coronaviridae. By contrast, no signal was
detected in 4 negative-control paired sera. The serum titers of
anti-hSARS antibodies of the tested patients are shown in Table
1.
1 TABLE 1 Name Date Lab No. Anti-SARS Patient A 25-Feb-03 S2728
<50 6-Mar-03 S2728 1600 Patient B 26-Feb-03 S2441 50 3-Mar-03
S2441 200 Patient C 4-Mar-03 S3279 200 14-Mar-03 S3279 1600 Patient
D 6-Mar-03 M41045 <50 11-Mar-03 MB943703 800 Patient E 4-Mar-03
M38953 <50 18-Mar-03 KWH03/3601 800 Control F 13-Feb-03 M27124
<50 1-Mar-03 MB942968 <50 Patient G 3-Mar-03 M38685 <50
7-Mar-03 KWH03/2900 Equivocal Blinded samples: 1a * Acute <50 1b
Convalescent 1600 2a * Acute 50 2b Convalescent >1600 3a * Acute
50 3b Convalescent >1600 4a * Acute <50 4b Convalescent
<50 5a * Acute <50 5b Convaelscent <50 6a * Acute <50
6b Convalescent <50 NB: * patients with SARS These results
indicated that this novel member of Coronaviridae is a key pathogen
in SARS.
[0194] 6.6 Sequences of the hSARS Virus
[0195] Total RNA from infected or uninfected FrHK-4 cells was
harvested two days post-infection. One-hundred ng of purified RNA
was reverse transcribed using Superscript II 10 reverse
transcriptase (Invitrogen) in a 20 .mu.l reaction mixture
containing 10 pg of a degenerated primer
(5'-GCCGGAGCTCTGCAGAATTCNNNNNNN-3', N=A, T, G or C: SEQ ID NO:5) as
recommended by the manufacturer. Reverse transcribed products were
then purified by a QIAquick PCR purification kit as instructed by
the manufacturer and eluted in 30 .mu.l of 10 mM Tris-HCl, pH 8.0.
Three .mu.l of purified cDNA products were add in a 25 .mu.l
reaction mixture containing 2.5 .mu.l of 10.times.PCR buffer, 4
.mu.l of 25 mM MgCl.sub.2, 0.5 .mu.l of 10 mM dNTP, 0.25 .mu.l of
AmpliTaq Gold.RTM. DNA polymerase (Applied Biosystems), 2.5 .mu.Ci
of [.alpha.-.sup.32P]CTP (Amersham), 2 .mu.l of 10 .mu.M primer
(5'-GCCGGAGCTCTGCAGAATT-C-3': SEQ ID NO:6). Reactions were thermal
cycled through the following profile: 94.degree. C. for 8 min
followed by 2 cycles of 94.degree. C. for 1 min, 40.degree. C. for
1 min, 72.degree. C. for 2 min. This temperature profile was
followed by 35 cycles of 94.degree. C. for 1 min, 60.degree. C. for
1 min, 72.degree. C. for 1 min. 6 .mu.l of the PCR products were
analyzed in a 5% denaturing polyacrylamide gel electrophoresis. Gel
was exposed to X-ray film and the film was developed after an
over-night exposure. Unique PCR products which were only identified
in infected cell samples were isolated from the gel and eluted in a
50 .mu.l of 1.times.TE buffer. Eluted PCR products were then
re-amplified in 25 .mu.l of reaction mixture containing 2.5 .mu.l
of 10.times.PCR buffer, 4 .mu.l of 25 mM MgCl.sub.2, 0.5 .mu.l ru
10 mM dNTP, 0.25 .mu.l of AmpliTaq Gold.RTM. DNA polymerase
(Applied Biosystems), 1 .mu.l of 10 .mu.M primer
(5'-GCCGGAGCTCTGCAGAATTC-3':SEQ ID NO:6). Reaction mixtures were
thermal cycled through the following profile: 94.degree. C. for 8
min followed by 35 cycles of 94.degree. C. for 1 min, 60.degree. C.
for 1 min, 72.degree. C. for 1 min. PCR products were cloned using
a TOPO TA cloning kit (Invitrogen) and ligated plasmids were
transformed into TOP10 E. coli competent cells (Invitrogen). PCR
inserts were sequenced by a BigDye cycle sequencing kit as
recommended by the manufacturer (Applied Biosystems) and sequencing
products were analyzed by an automatic sequencer (Applied
Biosystems, model number 3770). The obtained sequence (SEQ ID NO:1)
is shown in FIG. 1. The deducted amino acid sequence (SEQ ID NO:2)
from the obtained DNA sequence showed 57% homology to the
polymerase protein of identified coronaviruses.
[0196] Similarly, two other partial sequences (SEQ ID NOS:11 and
13) and deduced amino acid sequences (SEQ ID NOS:12 and 14,
respectively) were obtained from the hSARS virus and are shown in
FIGS. 8 (SEQ ID NOS:11 and 12) and 9 (SEQ ID NOS: 13 and 14).
[0197] The entire genomic sequence of hSARS virus is shown in FIG.
10 (SEQ ID NO:15). The deduced amino acid sequences of SEQ ID NO:15
in all three frames (SEQ ID NO:16, 240 and 737) are shown in FIG.
11 (SEQ ID NOS: 17-239, 241-736 and 738-1107). The deduced amino
acid sequences of the complement of SEQ ID NO:15 in all three
frames (SEQ ID NOS:1108, 1590 and 1965) are shown in FIG. 12 (SEQ
ID NOS:1109-1589, 1591-1964 and 1966-2470).
[0198] 6.7 Detection of hSARS Virus in Nasopharyngeal Aspirates
[0199] First, the nasopharyngeal aspirates (NPA) were examined by
rapid immunoflourescent antigen detection for influenza A and B,
parainfluenza types 1, 2 and 3, respiratory syncytial virus and
adenovirus (Chan K H, Maldeis N, Pope W, Yup A, Ozinskas A. Gill J,
Seto W H, Shortridge K F, Peiris J S M. Evaluation of Directigen
Fly A+B test for rapid diagnosis of influenza A and B virus
infections. J Clin Microbiol. 2002; 40: 1675-1680) and were
cultured for conventional respiratory pathogens on Mardin Darby
Canine Kidney, LLC-Mk2, RDE, Hep-2 and MRC-5 cells (Wiedbrauk D L,
Johnston S L G. Manual of clinical virology. Raven Press, New York.
1993). Subsequently, fetal rhesus kidney (FRhk-4) and A-549 cells
were added to the panel of cell lines used. Reverse transcription
polymerase chain reaction (RT-PCR) was performed directly on the
clinical specimen for influenza A (Fouchier R A, Bestebroer T M,
Herfst S, Van Der Kemp L, Rimmelzwan G F, Osterhaus A D. Detection
of influenza A virus from different species by PCR amplification of
conserved sequences in the matrix gene. J Clin Microbiol. 2000; 38:
4096-101) and human metapneumovirus (HMPV). The primers used for
HMPV were: for first round, 5'-AARGTSAATGCATCAGC-3' (SEQ ID NO. 7)
and 5'-CAKATTYTGCTTATGCTTTC-3' (SEQ ID NO:8); and nested primers:
5'-ACACCTGTTACAATACCAGC-3' (SEQ ID NO:9) and
5'-GACTTGAGTCCCAGCTCCA-3' (SEQ ID NO:10). The size of the nested
PCR product was 201 bp. An ELISA for mycoplasma was used to screen
cell cultures (Roche Diagnostics GmbH, Roche, Indianapolis,
USA).
[0200] RT-PCR Assay
[0201] Subsequent to culturing and genetic sequencing of the hSARS
virus from two patients (see Section 6.6, supra), an RT-PCR was
developed to detect the hSARS virus sequence from NPA samples.
Total RNA from clinical samples was reverse transcribed using
random hexamers and cDNA was amplified using primers
5'-TACACACCTCAGC-GTTG-3' (SEQ ID NO:3) and 5'-CACGAACGTGACGAAT-3'
(SEQ ID NO:4), which are constructed based on the in the presence
of 2.5 mM MgCl.sub.2 (94.degree. C. for 8 min followed by 40 cycles
of 94.degree. C. for 1 min, 50.degree. C. for 1 min, 72.degree. C.
for 1 min).
[0202] The summary of a typical RT-PCR protocol is as follows:
[0203] 1. RNA Extraction
[0204] RNA from 140 .mu.l of NPA samples is extracted by QIAquick
viral RNA extraction kit and is eluted in 50 .mu.l of elution
buffer.
2 2. Reverse transcription RNA 11.5 .mu.l 0.1 M DTT 2 .mu.l
5.times. buffer 4 .mu.l 10 mM dNTP 1 .mu.l Superscript II, 200
U/.mu.l (Invitrogen) 1 .mu.l Random hexamers, 0.3 .mu.g/.mu.l 0.5
.mu.l Reaction condition 42.degree. C., 50 min 94.degree. C., 3 min
4.degree. C.
[0205] 3. PCR
[0206] cDNA generated by random primers is amplified in a 50 ul
reaction as follows:
3 cDNA 2 .mu.l 10 mM dNTP 0.5 .mu.l 10.times. buffer 5 .mu.l 25 mM
MgCl.sub.2 5 .mu.l 25 .mu.M Forward primer 0.5 .mu.l 25 .mu.M
Reverse primer 0.5 .mu.l AmpliTaq Gold polymerase, 5 U/.mu.l 0.25
.mu.l (Applied Biosystems) Water 36.25 .mu.l
[0207] Thermal-cycle condition: 95.degree. C., 10 min, followed by
40 cycles of 95.degree. C., 1 min; 50.degree. C. 1 min; 72.degree.
C., 1 min.
[0208] 4. Primer Sequences
[0209] Primers were designed based on the RNA-dependent RNA
polymerase encoding sequence (SEQ ID NO:1) of the hSARS virus.
4 Forward primer: 5' TACACACCTCAGCGTTG 3' (SEQ ID NO:3) Reverse
primer: 5' CACGAACGTGACGAAT 3' (SEQ ID NO:4)
[0210] Product size: 182 bps
[0211] Real-Time Ouantitative PCR Assay
[0212] Total RNA from 140 .mu.l of nasopharyngeal aspirate (NPA)
was extracted by QIAamp.RTM. virus RNA mini kit (Qiagen) as
instructed by the manufacturer. Ten .mu.l of eluted RNA samples
were reverse transcribed by 200 U of Superscript II.RTM. reverse
transcriptase (Invitrogen) in a 20 .mu.l reaction mixture
containing 0.15 .mu.g of random hexamers, 10 mmol/l DTT, and 0.5
mmol/l dNTP, as instructed. Complementary DNA was then amplified in
a SYBR.RTM. Green I fluorescence reaction (Roche) mixtures.
Briefly, 20 .mu.l reaction mixtures containing 2 .mu.l of cDNA, 3.5
mmol/l MgCl.sub.2, 0.25 .mu.mol/l of forward primer
(5'-TACACACCTCAGCGTTG-3'; SEQ ID NO:3) and 0.25 .mu.mol/l reverse
primer (5'-CACGAACGTGACGAAT-3'; SEQ ID NO:4) were thermal-cycled by
a Light-Cycler (Roche) with the PCR program, [95.degree. C., 10 min
followed by 50 cycles of 95.degree. C., 10 min; 57.degree. C., 5
sec; 72.degree. C. 9 sec]. Plasmids containing the target sequence
were used as positive controls. Fluorescence signals from these
reactions were captured at the end of extension step in each cycle.
To determine the specificity of the assay, PCR products (184 base
pairs) were subjected to a melting curve analysis at the end of the
assay (65.degree. C. to 95.degree. C., 0.1.degree. C. per
second).
[0213] 6.8 Detection of N-Gene of hSARS Virus in Patients
[0214] 6.8.1 RT-PCR Diagnosis Protocol for Coronavirus in SARS
Patients
[0215] Equipment required (for 96 samples):
[0216] 1.times.SV Total RNA Isolation system
[0217] 2.times.Mega titer plate
[0218] 3.times.96-well PCR plate
[0219] 1.times.0.5-10 .mu.l multi-channel pipette
[0220] 1.times.10-100 .mu.l multi-channel pipette
[0221] 1.times.20-200 .mu.l multi-channel pipette
[0222] 1.times.vacuum pump
[0223] 1.times.swing-bucket rotor with microtest plate buckets
[0224] 2.times.PCR machine (96-well plate compatible)
[0225] 1.times.Gel electrophoresis apparatus
[0226] Station 1*--Clinical Samples Handling (1 Medical
Officer/Clinical Technician)
[0227] Aliquot 500 .mu.l sample in viral transport medium
(containing, per liter, 2 g of sodium bicarbonate, 5 g of bovine
serum albumin, 200 .mu.g of vancomycin, 18 .mu.g of amikacin, and
160 U of nystatin in Earle's balanced salt solution) from each
individual vial into a well of 96-well mega titer plate containing
500 .mu.l lysis buffer (1.times.) containing 100 .mu.l PK-15 cell
(ATCC CCL-33; 5.0.times.10.sup.5 cell/ml) in complete minimum
essential medium with Earle's salt (EMEM, Invirtogen) as internal
control.**
[0228] Mix the lysate by pipetting up-and-down 3 times
[0229] Proceed to Station 2.
[0230] *Station 1 should be carried out inside Class III biological
safety cabinet.
[0231] **At least two negative samples should be included in a
96-well platform as a negative control.
[0232] Station 2--Total RNA Extraction (1 Laboratory
Technician)
[0233] Set up the Vacuum Manifold unit. Place the binding plate
onto the Manifold Base.
[0234] Transfer the lysate from mega titer plate to each well of
the SV 96 Binding Plate (binding plate).
[0235] Apply vacuum until the lysate passes through the binding
plate. Release vacuum.
[0236] Add 500 .mu.l of SV RNA Wash Solution (wash solution) to
each well of binding plate.
[0237] Apply vacuum until the wash solution passes through binding
plate. Release vacuum.
[0238] Prepare DNase incubation mix for an entire 96-well plate as
below:
5 Yellow Core Buffer 2 ml 0.09 M MnCl.sub.2 250 .mu.l DNase I 250
.mu.l
[0239] Apply 25 .mu.l freshly prepared DNase incubation mix
directly to the membrane of the binding plate.
[0240] Incubate at 20-25.degree. C. for 10 minutes.
[0241] Add 200 pl of SV DNase Stop Solution to each well of the
binding plate.
[0242] Apply vacuum until the SV DNase Stop Solution passes through
the binding plate. Release vacuum.
[0243] Add 500 .mu.l wash solution to each well of the binding
plate.
[0244] Apply vacuum until wash solution passes through the binding
plate. Turn off vacuum.
[0245] Spin the binding plate at 3000.times.g for 30 seconds to
remove residue wash solution.
[0246] Transfer the binding plate on top of a 96-well RT plate.
[0247] Add 50 .mu.l nuclease-free water into each well of the
binding plate to elute RNA.
[0248] Incubate at room temperature for 1 minute.
[0249] Spin the binding plate at 3000.times.g, 4.degree. C. for 1
minute.
[0250] Collect eluted RNA in the 96-well RT plate.
[0251] Add 5 .mu.l of 3 M sodium acetate and 200 .mu.l of 95%
ethanol into each well of the plate.
[0252] Place the RT plate on ice and incubate for 15 minutes.
[0253] Spin the plate at 3000.times.g, 4.degree. C., 15
minutes.
[0254] Discard supernatant by inverting the plate and blotting on a
clean paper towel.
[0255] Wash the pellet with 200 .mu.l of 70% ethanol.
[0256] Spin the plate at 3000.times.g, 4.degree. C., 10
minutes.
[0257] Discard supernatant by inverting the plate and blotting on a
clean paper towel.
[0258] Air-dry the pellet for 5 minutes.
[0259] Add 12 .mu.l of nuclease-free water into each well.
[0260] Vortex the plate briefly to dissolve the pellet (for an
example result, see FIG. 18).
[0261] Proceed to Station 3.
[0262] Station 3--Reverse Transcription (1 Laboratory
Technician)
[0263] Prepare RT master mix for an entire 96-well plate in a
1.5-ml tube as below (100 reactions):
6 Per Reaction .times.100 Random hexamers, 3 .mu.g/.mu.l 0.05 .mu.l
5 .mu.l DNTPs, 10 mM 1 .mu.l 100 .mu.l First-strand buffer, 5x 4
.mu.l 400 .mu.l DTT, 0.1 M 2 .mu.l 200 .mu.l Superscript II, 200
U/.mu.l 1 .mu.l 100 .mu.l Total 8.05 .mu.l 805 .mu.l
[0264] Aliquot 100 .mu.l RT mix into 8 wells of a clean 96-well
master mix plate.
[0265] From this plate, transfer 8.05 .mu.l RT mix to each well of
RT plate containing 12 .mu.l RNA, mix by pipetting up-and-down for
3 times with a multi channel pipette. REPLACE TIP AFTER EACH
TRANSFER.
[0266] Incubate the samples at 42.degree. C. for 50 minutes
followed by 70.degree. C. for 15 minutes.
[0267] Proceed to Station 4.
[0268] Station 4--N-Gene Specific PCR (1 Laboratory Technician)
[0269] Prepare PCR master mix for an entire 96-well plate in two
2059 culture tubes as below (100 reactions):
7 N-specific PCR Control PCR Per 25 .mu.l Per 25 .mu.l Reaction
.times.100 Reaction .times.100 mQH.sub.2O 18.65 .mu.l 1865 .mu.l
17.65 .mu.l 1765 .mu.l 10.times. PCR buffer 2.5 .mu.l 250 .mu.l 2.5
.mu.l 250 .mu.l 25 mM MgC12 1.5 .mu.l 150 .mu.l 2.5 .mu.l 250 .mu.l
10 mM dNTPs 0.25 .mu.l 25 .mu.l 0.25 .mu.l 25 .mu.l Forward primer
0.5 .mu.l 50 .mu.l 0.5 .mu.l 50 .mu.l 10 .mu.M Reverse primer 0.5
.mu.l 50 .mu.l 0.5 .mu.l 50 .mu.l 10 .mu.M AmpliTaq Gold .RTM. 0.1
.mu.l 10 .mu.l 0.1 .mu.l 10 .mu.l 500 U Template DNA 1 .mu.l -- 1
.mu.l -- Total 25 .mu.l 2400 .mu.l 25 .mu.l 2400 .mu.l
[0270] N-gene specific PCR and control PCR are performed in two
individual PCR plates.
[0271] Aliquot 290 .mu.l PCR master mix into the first column of a
96-well PCR plate.
[0272] From the first column, aliquot 24 .mu.l of master mix into
each well of PCR plate.
[0273] Transfer 1 .mu.l of cDNA template (from station 4) into each
well of PCR plate.
[0274] Mix by pipetting up-and-down for 3 times with a
multi-channel pipette. REPLACE TIP AFTER EACH TRANSFER.
[0275] Seal the plate with sealing tape.
[0276] Perform the following reaction in two 96-well PCR
machines:
8 N-gene specific PCR Control PCR 94.degree. C. 10 minutes
94.degree. C. 10 minutes 94.degree. C. 30 seconds 94.degree. C. 30
seconds {close oversize brace} 40 cycles {close oversize brace} 35
cycles 56.degree. C. 30 seconds 55.degree. C. 30 seconds 72.degree.
C. 30 seconds 72.degree. C. 45 seconds 72.degree. C. 10 minutes
72.degree. C. 10 minutes
[0277] Station 5--Gel Electrophoresis (1 Laboratory Technician)
[0278] Mix 5 .mu.l of N-gene specific PCR product and 5 .mu.l
control PCR product with 1 .mu.l bromophenol blue loading dye
[0279] Load the samples into the wells of a 2% agarose gel.
[0280] Electrophoresize the PCR products at 140V, 250 mA for 30
minutes.
[0281] Stain the gel with ethidium bromide.
[0282] Visualize the products with UV and record the result.
[0283] 6.8.2 Using Primers of SEQ ID NOS:2480 and 2481
[0284] RT-PCR diagnostic protocol was performed as described in
Section 6.8.1 with some modifications.
[0285] RNA Isolation from Clinical Samples
[0286] Clinical samples including nasopharyngeal aspirates (NPA)
and stool specimens were provided by the Department of
Microbiology, The University of Hong Kong. In addition, tracheal
dispersion and lung biopsy from an index patient A described in New
Engl. J Med. 348:1967-76 (by Drosten C. S., et al., 2003) at three
time points was also collected. Sample collection was conducted
from Apr. 1 to Apr. 28, 2003 in local hospitals. Method of sample
collection was described in the previous section (also see, Poon et
al., 2003, Clinical Chemistry 49:953-955). Total RNA extraction
from clinical samples was carried out with SV96 Total RNA Isolation
System (Promega, Wis., USA), with following modifications from
manufacturer's protocol. Five-hundred (500) .mu.l of NPA/stool
sample in viral transport medium (containing, per liter, 2 g of
sodium bicarbonate, 5 g of bovine serum albumin, 200 .mu.g of
vancomycin, 18 .mu.g of amikacin, and 160 U of nystatin in Earle's
balanced salt solution) was mixed with equal volume of SV RNA Lysis
Buffer containing 100 .mu.l of pig kidney epithelial (PK-15) cell
(ATCC CCL-33; 5.0.times.10.sup.5 cells/ml) in complete minimum
essential medium with Earle's salt (EMEM, Invitrogen) as internal
control. The mixture was transferred to the wells of the SV 96
Binding Plate. After washing with 500 .mu.l of SV RNA Wash Solution
prior to elution step, the plate was spun at 3000.times.g for 30
seconds to remove residue wash solution. RNA was then eluted with
50 .mu.l of nuclease-free water, and was collected in a clean
96-well PCR plate by spinning the plate at 3000.times.g for 1
minute. Eluted RNA was then concentrated by incubating on ice for
15 minutes, in the presence of 5 .mu.l of 3 M sodium acetate and
200 .mu.l of 95% ethanol. After centrifugation at 3000.times.g,
4.degree. C. for 15 minutes, RNA pellet was washed with 200 .mu.l
of 75% ethanol and dissolved with 12 .mu.l of nuclease-free water.
Extracted RNA was immediately reverse-transcribed to first-strand
cDNA.
[0287] First-Strand cDNA Synthesis
[0288] Reverse-transcription was performed with 200 U of
Superscript.RTM. II reverse transcriptase (Invitrogen, USA) in a 20
.mu.l reaction containing 0.15 .mu.g of random hexamers, RT buffer
(1.times.), 10 mM dithiothreitol (DTT) and 0.5 mM deoxynucleotide
triphosphates (dNTPs). Reaction was carried out in Peltier Thermal
Cycler (MJ Research) with the following conditions: 50 minutes at
42.degree. C. followed by 15 minutes at 70.degree. C.
[0289] Polymerase Chain Reaction (PCR)
[0290] Primers were designed according to complete SARS CoV genomic
sequence of a local specimen HK-39 announced previously (accession
no. AY278491). Forward primer (SRS251: 5'-GCAGTCAAGCCTCTTCTCG-3';
SEQ ID NO:2480, corresponding to nt 28658-28676 of HK-39 SARS
genome, i.e., CCTCC200303) and reverse primer (SRS252:
5'-GCCTCAGCAGCAGATTTC-3', SEQ ID NO:2481; corresponding to nt
28866-28883 of HK-39 SARS genome) amplified a 225 bp fragment from
the region of N-gene that showed no homology to other coronavirus.
Primers amplifying RNA-dependent RNA polymerase (1b gene) were used
as parallel control (coro3: 5'-TACACACCTCAGCGTTG-3' (SEQ ID NO:3),
corresponding to nt 18041-18057; and coro4: 5'-CACGAACGTGACGAAT-3'
(SEQ ID NO:4), corresponding to nt 18207-18222, Department of
Microbiology, the University of Hong Kong). Both amplicons were
cloned into same pCR2.1 cloning vector (FIG. 17). Serially diluted
plasmid was then used to determine the dynamic range and optimal
condition of the PCRs (FIGS. 21A and 21B). Another set of primer
that amplifying a 745 bp fragment from pig .beta.-actin gene was
employed as an internal control for the diagnostic PCR assay
(actin-F: 5'-TGAGACCTTCAACACGCC-3'(SEQ ID NO:2482); and actin-R:
5'-ATCTGCTGGAAGGTGGAC-3' (SEQ ID NO:2483)).
[0291] Conventional PCR and gel electrophoresis was carrid out as
preliminary experiment. Briefly, 1 .mu.l of cDNA from clinical
samples was amplified with 0.5 U Taq DNA polymerase recombinant
(Invitrogen Life Technologies) in a 25-.mu.l reaction containing
PCR buffer (1.times.), 1.5 mM MgCl.sub.2, 0.1 mM dNTPs and 0.5 pmol
of each forward and reverse primers. Reaction was performed in
Peltier Thermal Cycler (MJ Research) with the following conditions:
3 minutes at 94.degree. C., followed by 50 cycles of 94.degree. C.
for 10 seconds, 56.degree. C. for 10 seconds, 72.degree. C. for 10
seconds, and a 10-minute final extension step at 72.degree. C.
Amplicons were analyzed with 2% agarose gel electrophoresis (FIG.
23). Quantitative real-time PCR using SYBR.RTM. SYBR.RTM. green
fluorophore was performed in diagnosis of clinical samples. In a 25
.mu.l reaction, 1 .mu.l cDNA template was mixed with 12.5 .mu.l
(2.times.) Green PCR Master Mix (Applied Biosystems) and 0.5 pmol
of each forward and reverse primer. Volume of the reaction was
adjusted to 25 .mu.l with distilled water. Reactions were performed
in the iCycler iQ Real-Time PCR Detection System (Bio-Rad) under
the same condition as the conventional PCR. Fluorescence signals
(FAM, excitation=490 nm, emission=530 nm) were collected at the end
of each extension step during the PCR cycles (FIG. 22A). Threshold
cycle (Ct) of each sample was determined using maximum curvature
approach. Melting curve analysis was performed after 10 minutes
final extension (FIG. 22B). cDNA from non-SARS patients, including
patients suffering from adenovirus (n=5), repiratory syncytial
virus (n=5), human metapneumovirus (n=5), influenza A virus (n=5),
or influenza B virus (n=5) infection, were used as negative
controls for the assay.
[0292] Northern Blot Analysis
[0293] SARS-CoV HK-39 strain infected Vero cell was provided by
Department of Microbiology, the University of Hong Kong. Total RNA
was extracted from the cell with TRIzol.degree. reagent (Invitrogen
Life Technologies) according to the manufacturer's protocol. Eight
(8) .mu.g of total RNA was separated by electrophoresis on a 1%
agarose gel containing 3.7% formaldehyde. RNA was transferred to a
positively charged nylon membrane Roche Diagnostic Corporation) by
capillary blotting and fixed by UV cross-linking. cDNA synthesized
with the same RNA sample was used as template for probe synthesis.
Four pairs of primers amplifying fragments from 1b (nt 18057-18222;
SEQ ID NO:2484), S (nt 21920-22107; SEQ ID NO:2485), M (nt
26867-26996; SEQ ID NO:2486) and N (nt 28658-28883; SEQ ID NO:2487)
gene were used in probe synthesis. DIG-labeling of probes,
hybridization and detection of bands were performed with the
digoxigenin system according to the manufacturer's procedures
(Roche Molecular Biochemcials). Signals were then analysed with
chemiluminescence (FIG. 24).
[0294] Results and Discussion
[0295] A large-scale RT-PCR assay provides a rapid means in
monitoring and screening of SARS suspects. The result can be used
to complement clinical diagnostic evaluation. In order to achieve a
diagnostic purpose, the assay should be reliable and its accuracy
should be assured so as to prevent occurrence of both false
negative and false positive results. However, accuracy of the test
may be influenced by several factors. A common technical problem
with PCR is a failure of amplification due to the presence of PCR
inhibitors (see FIG. 21).
[0296] These PCR inhibitors included heme compounds found in blood,
aqueous and vitreous humors, heparin, EDTA, urine and polyamines
(Fredricks et al., 1998, J. Clin. Micro. 36:2810-16). Currently,
NPA or stool samples were collected into transport medium to
maintain the viability of the viral particles. RT-PCR was inhibited
when total RNA extracted was used directly for first-strand cDNA
synthesis without any treatment (25 out of 27 samples) in
preliminary experiment. However, after a simple ethanol
precipitation step, the amplification of DNA could be retained
(FIG. 19). Same result was obtained by either using SV or SV96
total RNA Isolation System (data not shown). It demonstrated that
some components either in the medium or NPA/stool samples would
affect the downstream processes of the diagnosis test.
[0297] In addition, current sample collection procedure dilutes the
virus titer in the samples, especially during early stage of
infection, in which the virus titer is low in nasal and throat swab
specimens (Drosten et al., 2003, New England Journal of Medicine,
on-line at http://content.nejm.org/cgi/reprint/NEJMoa030747v2). It
was suggested that the sensitivity of PCR tests for SARS depended
on the quality of the specimen and the time of testing during the
course of the illness. In order to increase sensitivity of the
test, total RNA isolated from clinical samples was concentrated
prior to 1st strand cDNA synthesis.
[0298] In order to avoid false negative PCR results due to failure
in the process of RNA isolation and 1st strand cDNA synthesis,
total RNA was extracted from clinical samples in parallel with
PK-15 mammalian cells. FIG. 23 showed the RT-PCR screening result
on 48 clinical samples, including both NPA and stool samples.
Diagnostic PCR was performed in parallel with .beta.-actin PCR. All
samples were positive in .beta.-actin PCR. The result indicated
that RNA and cDNA could be extracted and synthesized successfully
from the samples in a single-step protocol as disclosed herein.
With this internal control, total RNA isolation and cDNA synthesis
from the samples were ensured, which eliminated false negative that
resulted from failure in either one of the above processes.
Moreover, 96-well assay format currently developed can be adopted
into a high-throughput screening protocol, with which we are able
to obtain diagnostic result of more than 90 clinical samples in 3
hours with 1 clinical personnel, while the current existing
protocol, in which samples are proceeded in individual tubes, can
only handle about 30-50 samples a day per technician.
[0299] Real-time quantitative PCR assay is more sensitive than
conventional agarose gel-electrophoresis-associated PCR assay (Poon
et al., J. Clin. Virol. 28:233-8) and therefore employed for
SARS-CoV diagnosis purpose. Positive signals were detected in 38 of
136 randomly selected clinical samples in both N-gene and 1b-gene
specific PCR. Among these 38 positives, 3 were stool samples (2.2%)
and 35 were NPA samples (25.7%). Detection rate of the assay
employing N-gene specific RT-PCR at different time points was shown
in Table 2.
9TABLE 2 Date of onset No. of sample No. of positive Detection rate
(%) 1-2 15 2 13.3 3-4 17 4 23.5 5-6 15 4 26.7 7-8 13 5 38.5 9-10 9
4 44.4 Negative control 19 All negative --
[0300] Affirmative of these 38 positive cases was confirmed by
melting curve analysis of PCR products. Specific melting
temperature of N gene and 1b gene PCR products (85.5.degree. C. and
80.5.degree. C., respectively) indicated that the target framgments
were amplified in the reaction. Specificity of the assay was also
validated with non-SARS patients samples, including patients
suffering from adenovirus (n=5), repiratory syncytial virus (n=5),
human metapneumovirus (n=5), influenza A virus (n=5) and influenza
B virus (n=5). The result shows that all of these samples were
negative in the assay (Fig. ??). These results indicate that the
N-gene specific RT-PCR assay is specific for SARS-CoV
diagnosis.
[0301] Furthermore, we also demonstrated that the N-gene specific
PCR was more sensitive than that of PCR amplifying 1b RNA
polymerase gene. Amplification conditions for both PCR assays were
optimized (see FIG. 22) first with the plasmid construct containing
1:1 ratio of 1b- and N-gene fragment (see FIG. 20). Dynamic range
of N-gene specific PCR was obtained (Fig. ??) and it was found to
be with lower Ct values than that of 1b-specific PCR. This revealed
that N-gene specific PCR could achive higher amplification
efficiency than 1b-gene specific PCR when using same copy number of
template. PCR with cDNA from clinical samples or virus infected
Vero cells were then performed. FIG. 22A shows the Ct and
half-maximal values of the fluorescent signal of N gene and 1b gene
specific PCR generated from NPA, tracheal dispersion and lung
biopsy from patient A. The results indicated that fluorescent
signals given in N gene specific PCR are higher (26.0% in average,
ranged from 6.3-60%) than that of 1b specific PCR in all positive
samples. Furthermore, Ct values of N gene specific PCR are lower
(0.1-4.6 cycles) than that of 1b specific PCR among most of the
SARS-CoV positive samples (Table 3).
10TABLE 3 S/N N lb ?Ct S/N N lb ?Ct S/N N lb ?Ct S/N N lb ?Ct 56851
27.1 27.8 0.6 34862 31.9 33.2 1.3 67429 35.3 35.6 0.3 68796 32.4
34.5 2.1 55751 27.6 27.7 0.1 45971 43.6 48.2 4.6 67438 28.4 27.5
-0.9 68798 28.8 28.4 -0.4 62290 41.9 43.2 1.3 45972 43.6 46.5 2.9
68116 30.0 33.5 3.5 68800 34.6 38.3 3.7 55531 41.0 43.1 2.1 45973
42.1 43.2 1.1 68118 36.7 37.7 1.0 68801 31.9 32.8 0.9 55963 41.7
42.3 0.6 69145 27.2 27.7 0.5 68134 32.4 33.2 0.8 70562 40.2 43.3
3.1 65733 43.6 44.9 1.3 56386 32.8 33.8 1.0 68184 30.6 32.4 1.8
70589 35.5 38.2 2.7 34862 33.5 33.7 0.2 55527 37.3 39.4 2.1 68185
27.5 30.1 2.6 70591 36.0 38.2 2.2 32814 38.6 40.8 2.2 56851 23.9
26.1 2.2 68187 40.3 41.5 1.2 70059 41.4 43.1 1.7 33935 35.3 36.5
1.2 69073 24.3 26.1 1.3 68788 35.5 37.6 2.1 34861 31.4 31.7 0.3
67423 28.7 29.4 0.7 68791 34.8 35.3 0.5 .DELTA.Ct = 1.49 .+-. 0.47,
95% confidence intervals = 0.74 to 2.23 (F-test)
[0302] Statistic analysis indicates that Ct of N-gene PCR assay is
significantly lower than that of ab-gene assay (95% confidence
interval=0.74 to 2.23, F-test). Stronger fluorescence signals and
lower Ct values of N gene specific PCR provide a more sensitive
diagnostic result and much target for the assay.
[0303] Using cDNA from SARS-CoV infected Vero cells, amplification
curves shown in FIG. 21B show the differences between N gene and 1b
gene specific PCR. Ct of the N gene and 1b gene specific PCR was
35.3 and 37.8, respectively. This phenomenon had two main causes:
(1) Expression level of N gene was higher than that of 1b gene;
and; (2) Copy number of N gene was much larger than that of 1b gene
because each transcript preceded a copy of N gene, in SARS-CoV
infected cells. Northern blot analysis supported this hypothesis
(FIG. 24). When N-gene specific PCR product was used as a probe, at
least five transcripts from the virus were hybridized and gave
positive signals (FIG. 24). This result agreed with the findings in
which five subgenomic mRNAs were detected by Northern hybridization
of RNA from SARS-CoV infected cells using a probe derived from the
3' untranslated region (Rota et al., 2003, Science 300:1394-99). On
the other hand, when 1b PCR product was used as a probe, only 2
transcripts with high molecular size were hybridized, demonstrating
that the copy number of N gene was much higher than that of 1b
gene, during transcription and gene expression in the host cells.
The Northern hybridization result strongly supports the conclusion
that PCR amplifying regions in N gene of the SARS-CoV are more
sensitive than other regions as a target for diagnostic screening.
It is possible that amplification of more than one genome region
may increase the specificity of the test (Yam W. C., et al., 2003,
J. Clin. Microbiol. 41:4521-24).
[0304] In conclusion, we have developed a new generation of RT-PCR
diagnosis test which is more sensitive than conventional diagnostic
test for the detection of the coronavirus associated with SARS. The
assay provides a high throughput, highly sensitive screening
platform, which enables us to scale up to test hundreds of
thousands of suspected SARS cases each day in a single working
line. Incorporation of PK-15 cell as an internal control in the
assay and use of N gene as a diagnosis locus in addition to 1b gene
can enhance the sensitivity and accuracy of the test. We are
adapting the protocol to 96-well real-time quantitative PCR and
sequencing format to shorten the time required for the test and to
obtain information on genotypic variation of the virus.
[0305] Clinical Results
[0306] Clinical Findings:
[0307] All 50 patients with SARS were ethnic Chinese. They
represented 5 different epidemiologically linked clusters as well
as additional sporadic cases fitting the case definition. They were
hospitalized at a mean of 5 days after the onset of symptoms. The
median age was 42 years (range of 23 to 74) and the female to male
ratio was 1.3. Fourteen (28%) were health care workers and five
(10%) had a history of visit to a hospital experiencing a major
outbreak of SARS. Thirteen (26%) patients had household contacts
and 12 (24%) others had social contacts with patients with SARS.
Four (8%) had a history of recent travel to mainland China.
[0308] The major complaints from most patients were fever (90%) and
shortness of breath. Cough and myalgia were present in more than
half the patients (Table 4). Upper respiratory tract symptoms such
as rhinorrhea (24%) and sore throat (20%) were present in a
minority of patients. Diarrhea (10%) and anorexia (10%) were also
reported. At initial examination, auscultatory findings, such as
crepitations and decreased air entry, were present in only 38% of
patients. Dry cough was reported by 62% of patients. All patients
had radiological evidence of consolidation, at the time of
admission, involving 1 zone (in 36), 2 zones (13) and 3 zones
(1).
11 TABLE 4 Clinical symptoms Number (percentage) Fever 50 (100%)
Chill or rigors 37 (74%) Cough 31 (62%) Myalgia 27 (54%) Malaise 25
(50%) Running nose 12 (24%) Sore throat 10 (20%) Shortness of
breath 10 (20%) Anorexia 10 (20%) Diarrhea 5 (10%) Headache 10
(20%) Dizziness 6 (12%) * Truncal maculopapular rash was noted in 1
patient.
[0309] In spite of the high fever, most patients (98%) had no
evidence of a leukocytosis. Lymphopenia (68%), leucopenia (26%),
thrombocytopenia (40%) and anemia (18%) were present in peripheral
blood examination (Table 5). Parenchymal liver enzyme, alanine
aminotransferase (ALT) and muscle enzyme, creatinine kinase (CPK)
were elevated in 34% and 26% respectively.
12TABLE 5 Laboratory Percentage parameter Mean (range) of abnormal
Normal range Haemoglobin 12.9 (8.9-15.9) 11.5-16.5 g/dl Anaemia 9
(18%) White cell count 5.17 (1.1-11.4) 4-11 .times. 10.sup.9/L
Leucopenia 13 (26%) Lymphocyte count 0.78 (0.3-1.5) 1.5-4.0 .times.
10.sup.9/L Significant 34 (68%) lymphopenia (<1.0 .times.
10.sup.9/L) Platelet count 174 (88-351) 150-400 .times. 10.sup.9/L
Thrombocytopenia 20 (40%) Alanine amino- 63 (11-350) 6-53 U/L
transaminase (ALT) Elevated ALT 17 (34%) Albumin 37 (26-50) 42-54
g/L Low albumin 34 (68%) Globulin 33 (21-42) 24-36 g/L Elevated
globulin 10 (20%) Creatinine kinase 244 (31-1379) 34-138 U/L
Elevated creat- 13 (26%) inine kinase
[0310] Routine microbiological investigations for known viruses and
bacteria by culture, antigen detection, and PCR were negative in
most cases. Blood culture was positive for Escherichia coli in a
74-year-old male patient, who was admitted to intensive care unit,
and was attributed to hospital acquired urinary tract infection.
Klebsiella pneumoniae and Hemophilus influenzae were isolated from
the sputum specimens of 2 other patients on admission.
[0311] Oral levofloxacin 500 mg q24h was given in 9 patients and
intravenous (1.2 g q8h)/oral (375 mg tid) amoxicillin-clavulanate
and intravenous/oral clarithromycin 500 mg q12h were given in
another 40 patients. Four patients were given oral oseltamivir 75
mg bid. In one patient, intravenous ceftriaxone 2 gm q24h, oral
azithromycin 500 mg q24h, and oral amantadine 100 mg bid were given
for empirical coverage of typical and atypical pneumonia.
[0312] Nineteen patients progressed to severe disease with oxygen
desaturation and were required intensive care and ventilatory
support. The mean number of days of deterioration from the onset of
symptoms was 8.3 days. Intravenous ribavirin 8 mg/kg q8h and
steroid was given in 49 patients at a mean day of 6.7 after onset
of symptoms.
[0313] The risk factors associated with severe complicated disease
requiring intensive care and ventilatory support were older age,
lymphopenia, impaired ALT, and delayed initiation of ribavirin and
steroid (Table 6). All the complicated cases were treated with
ribavirin and steroid after admission to the intensive care unit
whereas all the uncomplicated cases were started on ribavirin and
steroid in the general ward. As expected, 31 uncomplicated cases
recovered or improved whereas 8 complicated cases deteriorated with
one death at the time of writing. All 50 patients were monitored
for a mean of 12 days at the time of writing.
13 TABLE 6 Complicated Uncomplicated case case (n = 19) (n =31) P
value Mean (SD) age (range) 49.5 .+-. 12.7 39.0 .+-. 10.7 P <
0.01 Male/Female ratio 8/11 14/17 N.S. Underlying illness 5
.sup..dagger. 1 .sup..dagger-dbl. P < 0.05 Mode of contact
Travel to China 1 3 N.S. Health care worker 5 9 N.S. Hospital visit
1 4 N.S. Household contact 8 5 P < 0.05 Social contact 4 10 N.S.
Mean (SD) duration of 5.2 .+-. 2.0 4.7 .+-. 2.5 N.S. symptoms to
admission (days) Mean (SD) admission 38.8 .+-. 0.9 38.7 .+-. 0.8
N.S. temperature (.degree. C.) Mean (SD) initial total 5.1 .+-. 2.4
5.2 .+-. 1.8 N.S. peripheral WBC count (.times.10.sup.9/L) Mean
(SD) initial lympho- 0.66 .+-. 0.3 0.85 .+-. 0.3 P < 0.05 cyte
count (.times.10.sup.9/L) Presence of thrombo- 8 12 N.S. cytopenia
(<150 .times. 10.sup.9/L) Impaired liver function 11 6 P <
0.01 test CXR changes (number of 1.4 1.2 N.S. zone affected) Mean
(SD) day of deteri- 8.3 .+-. 2.6 Not applicable oration from the
onset of symptoms .sctn. Mean (SD) day of initia- 7.7 .+-. 2.9 5.7
.+-. 2.6 P < 0.05 tion of Ribavirin & steroid from the onset
of symptoms Initiation of ribavirin 12 0 P < 0.001 & steroid
after deteri- oration Response to ribavirin 11 28 P < 0.05 &
steroid Outcome Improved or recovered 10 31 P < 0.01 Not
improving .parallel. 8 0 P < 0.01 * Multi-variant analysis is
not performed due to low number of cases; .sup..dagger.2 patients
had diabetic mellitus, 1 had hypertrophic ostructive
cardiomyopathy, 1 had chronic active hepatitis B, and 1 had brain
tumour; .sup..dagger-dbl.1 patient had essential hypertension;
.sctn. desaturation requiring intensive care support; .parallel. 1
died.
[0314] Two virus isolates, subsequently identified as a member of
Coronaviridae (see below), were isolated from two patients. One was
from an open lung biopsy tissue of a 53-year-old Hong Kong Chinese
resident and the other from a nasopharyngeal aspirate of a 42
year-old female with good previous health. The 53-year old male had
a history of 10-hour household contact with a Chinese visitor who
came from Guangzhou and later died from SARS. Two days after this
exposure, he presented with fever, malaise, myalgia, and headache.
Crepitations were present over the right lower zone and there was a
corresponding alevolar shadow on the chest radiograph.
Hematological investigation revealed lymphopenia of
0.7.times.10.sup.9/l with normal total white cell and platelet
counts. Both ALT (41 U/L) and CPK (405 U/L) were impaired. Despite
a combination of oral azithromycin, amantadine, and intravenous
ceftriaxone, there was increasing bilateral pulmonary infiltrates
and progressive oxygen desaturation. Therefore, an open lung biopsy
was performed 9 days after admission. Histopathological examination
showed a mild interstitial inflammation with scattered alveolar
pneumocytes showing cytomegaly, granular amphophilic cytoplasm and
enlarged nuclei with prominent nucleoli. No cells showed inclusions
typical of herpesvirus or adenovirus infection. The patient
required ventilation and intensive care after the operative
procedure. Empirical intravenous ribavirin and hydrocortisone were
given. He succumbed 20 days after admission. In retrospect,
coronavirus-like RNA was detected in his nasopharyngeal aspirate,
lung biopsy and post-mortem lung. He had a significant rise in
titer of antibodies against his own hSARS isolate from 1/200 to
1/1600.
[0315] The second patient from whom a hSARS virus was isolated, was
a 42-year-old female with good past health. She had a history of
travel to Guangzhou in mainland China for 2 days. She presented
with fever and diarrhea 5 days after her return to Hong Kong.
Physical examination showed crepitation over the right lower zone
which had a corresponding alveolar shadow on the chest radiograph.
Investigation revealed leucopenia (2.7.times.10.sup.9/L),
lymphopenia (0.6.times.10.sup.9/L), and thrombocytopenia
(104.times.10.sup.9/L). Despite the empirical antimicrobial
coverage with amoxicillin-clavulanate, clarithromycin, and
oseltamivir, she deteriorated 5 days after admission and required
mechanical ventilation and intensive care for 5 days. She gradually
improved without receiving treatment with ribavirin or steroid. Her
nasopharyngeal aspirate was positive for the virus in the RT-PCR
and she was seroconverted from antibody titre <1/50 to 1/1600
against the hSARS isolate.
[0316] Virological Findings:
[0317] Viruses were isolated on FRhk-4 cells from the lung biopsy
and nasopharyngeal aspirate respectively, of two patients described
above. The initial cytopathic effect appeared between 2 and 4 days
after inoculation, but on subsequent passage, cytopathic effect
appeared in 24 hours. Both virus isolates did not react with the
routine panel of reagents used to identify virus isolates including
those for influenza A, B parainfluenza types 1,2,3, adenovirus and
respiratory syncytial virus (DAKO, Glostrup, Denmark). They also
failed to react in RT-PCR assays for influenza A and HMPV or in PCR
assays for mycoplasma. The virus was ether sensitive, indicating
that it was an enveloped virus. Electron microscopy of negatively
stained (2% potassium phospho-tungstate, pH 7.0) cell culture
extracts obtained by ultracentrifugation showed the presence of
pleomorphic enveloped viral particles, of about 80-90 nm (ranging
70-130 nm) in diameter, whose surface morphology appeared
comparable to members of Coronaviridae (FIG. 5a). Thin section
electron microscopy of infected cells revealed virus particles of
55-90 nm diameter within the smooth-walled vesicles in the
cytoplasm (FIG. 5b). Virus particles were also seen at the cell
surface. The overall findings were compatible with infections in
the cells caused by viruses of Coronaviridae.
[0318] A thin section electron micrograph of the lung biopsy of the
53 year old male contained 60-90-nm viral particles in the
cytoplasm of desquamated cells. These viral particles were similar
in size and morphology to those observed in the cell-cultured virus
isolate from both patients (FIG. 4).
[0319] The RT-PCR products generated in a random primer RT-PCR
assay were analyzed and unique bands found in the virus infected
specimen was cloned and sequenced. Of 30 clones examined, a clone
containing 646 base pairs (SEQ ID NO:1) of unknown origin was
identified. Sequence analysis of this DNA fragment suggested this
sequence had a weak homology to viruses of the family of
Coronaviridae (data not shown). Deducted amino acid sequence (215
amino acids: SEQ ID NO:2) from this unknown sequence, however, had
the highest homology (57%) to the RNA polymerase of bovine
coronavirus and murine hepatitis virus, confirming that this virus
belongs to the family of Coronaviridae. Phylogenetic analysis of
the protein sequences showed that this virus, though most closely
related to the group II coronaviruses, was a distinct virus (FIGS.
5a and 5b).
[0320] Based on the 646 bp sequence of the isolate, specific
primers for detecting the new virus was designed for RT-PCR
detection of this hSARS virus genome in clinical specimens. Of the
44 nasopharyngeal specimens available from the 50 SARS patients, 22
had evidence of hSARS RNA. Viral RNA was detectable in 10 of 18
fecal samples tested. The specificity of the RT-PCR reaction was
confirmed by sequencing selected positive RT-PCR amplified
products. None of 40 nasophararyngeal and fecal specimens from
patients with unrelated diseases were reactive in the RT-PCR
assay.
[0321] To determine the dynamic range of real-time quantitative
PCR, serial dilutions of plasmid DNA containing the target sequence
were made and subjected to the real-time quantitative PCR assay. As
shown in FIG. 7A, the assay was able to detect as little as 10
copies of the target sequence. By contrast, no signal was observed
in the water control (FIG. 7A). Positive signals were observed in
23 out of 29 serologically confirmed SARS patients. In all of these
positive cases, a unique PCR product (T.sub.m=82.degree. C.)
corresponds to the signal from the positive control was observed
(FIG. 7B, and data not shown). These results indicated this assay
is highly specific to the target. The copy numbers of the target
sequence in these reactions range from 4539 to less than 10. Thus,
as high as 6.48.times.10.sup.5 copies of this viral sequence could
be found in 1 ml of NPA sample. In 5 of the above positive cases,
it was possible to collect NPA samples before seroconvertion. Viral
RNA was detected in 3 of these samples, indicating that this assay
can detect the virus even at the early onset of infection.
[0322] To further validate the specificity of this assay, NPA
samples from healthy individuals (n=11) and patients suffered from
adenovirus (n11), respiratory syncytial virus (n=11), human
metapneumovirus (n=11), influenza A virus (n=13) or influenza B
virus (n=1) infection were recruited as negative controls. All of
these samples, except one, were negative in the assay. The false
positive case was negative in a subsequence test. Taken together,
including the initial false positive case, the real-time
quantitative PCR assay has sensitivity of 79% and specificity of
98%.
[0323] Epidemiological data suggest that droplet transmission is
one of the major route of transmission of this virus. The detection
of live virus and the detection of high copies of viral sequence
from NPA samples in the current study clearly support that cough
and sneeze droplets from SARS patients might be the major source of
this infectious agent. Interestingly, 2 out of 4 available stool
samples form the SARA patients in this study were positive in the
assay (data not shown). The detection of the virus in feces
suggests that there might be other routes of transmission. It is
relevant to note that a number of animal coronaviruses are spread
via the fecal-oral route (McIntosh K., 1974, Coronaviruses: a
comparative review. Current Top Microbiol Immunol. 63: 85-112).
However, further studies are required to test whether the virus in
feces is infectious or not.
[0324] Currently, apart form this hSARS virus, there are two known
serogroups of human coronaviruses (229E and OC43) (Hruskova J. et
al., 1990, Antibodies to human coronaviruses 229E and OC43 in the
population of C. R., Acta Virol. 34:346-52). The primer set used in
the present assay does not have homology to the strain 229E. Due to
the lack of available corresponding OC43 sequence in the Genebank,
it is not known whether these primers would cross-react with this
strain. However, sequence analyses of available sequences in other
regions of OC43 polymerase gene indicate that the novel human virus
associated with SARS is genetically distinct from OC43.
Furthermore, the primers used in this study do not have homology to
any of sequences from known coronaviruses. Thus, it is very
unlikely that these primers would cross-react with the strain
OC43.
[0325] Apart from the novel pathogen, metapneumovirus was reported
to be identified in some of SARS patients (Center for Disease
Control and Prevention, 2003, Morbidity and Mortality Weekly Report
52: 269-272). No evidence of metapneumovirus infection was detected
in any of the patients in this study (data not shown), suggesting
that the novel hSARS virus of the invention is the key player in
the pathogenesis of SARS.
[0326] Immunofluorescent Antibody Detection:
[0327] Thirty-five of the 50 most recent serum samples from
patients with SARS had evidence of antibodies to the hSARS (see
FIG. 3). Of 27 patients from whom paired acute and convalescent
sera were available, all were seroconverted or had >4 fold
increase in antibody titer to the virus. Five other pairs of sera
from additional SARS patients from clusters outside this study
group were also tested to provide a wider sampling of SARS patients
in the community and all of them were seroconverted. None of 80
sera from patients with respiratory or other diseases as well as
none of 200 normal blood donors had detectable antibody.
[0328] When either seropositivity to HP-CV in a single serum or
viral RNA detection in the NPA or stool are considered evidence of
infection with the hSARS, 45 of the 50 patients had evidence of
infection. Of the 5 patients without any virological evidence of
Coronaviridae viral infection, only one of these patients had their
sera tested >14 days after onset of clinical disease.
[0329] Discussion
[0330] The outbreak of SARS is unusual in a number of aspects, in
particular, in the appearance of clusters of patients with
pneumonia in health care workers and family contacts. In this
series of patients with SARS, investigations for conventional
pathogens of atypical pneumonia proved negative. However, a virus
that belongs to the family Coronaviridae was isolated from the lung
biopsy and nasopharyngeal aspirate obtained from two SARS patients,
respectively. Phylogenetically, the virus was not closely related
to any known human or animal coronavirus or torovirus. The present
analysis is based on a 646 bp fragment (SEQ ID NO:1) of the
polymerase gene, which indicates that the virus relates to
antigenic group 2 of the coronaviruses along with murine hepatitis
virus and bovine coronavirus. However, viruses of the Coronaviridae
can undergo heterologous recombination within the virus family and
genetic analysis of other parts of the genome needs to be carried
out before the nature of this new virus is more conclusively
defined (Holmes K V. Coronaviruses. Eds Knipe D M, Howley P M
Fields Virology, 4th Edition, Lippincott Williams & Wilkins,
Philadelphia, 1187-1203). The biological, genetic and clinical
data, taken together, indicate that the new virus is not one of the
two known human coronaviruses.
[0331] The majority (90%) of patients with clinically defined SARS
had either serological or RT-PCR evidence of infection by this
virus. In contrast, neither antibody nor viral RNA was detectable
in healthy controls. All 27 patients from whom acute and
convalescent sera were available demonstrated rising antibody
titers to hSARS virus, strengthening the contention that a recent
infection with this virus is a necessary factor in the evolution of
SARS. In addition, all five pairs of acute and convalescent sera
tested from patients from other hospitals in Hong Kong also showed
seroconversion to the virus. The five patients who has not shown
serological or virological evidence of hSARS virus infection, need
to have later convalescent sera tested to define if they are also
seroconverted. However, the concordance of the hSARS virus with the
clinical definition of SARS appears remarkable, given that clinical
case definitions are never perfect.
[0332] No evidence of HMPV infection, either by RT-PCR or rising
antibody titer against HMPV, was detected in any of these patients.
No other pathogen was consistently detected in our group of
patients with SARS. It is therefore highly likely that that this
hSARS virus is either the cause of SARS or a necessary
pre-requisite for disease progression. Whether or not other
microbial or other co-factors play a role in progression of the
disease remains to be investigated.
[0333] The family Coronaviridae includes the genus Coronavirus and
Torovirus. They are enveloped RNA viruses which cause disease in
humans and animals. The previously known human coronaviruses, types
229E and OC43 are the major causes of the common cold (Holmes K V.
Coronaviruses. Eds Knipe D M, Howley P M Fields Virology, 4th
Edition, Lippincott Williams & Wilkins, Philadelphia, pp.
1187-1203). But, while they can occasionally cause pneumonia in
older adults, neonates or immunocompromised patient (El-Sahly H M,
Atmar R L, Glezen W P, Greenberg S B. Spectrum of clinical illness
in hospitalizied patients with "common cold" virus infections. Clin
Infect Dis. 2000; 31: 96-100; and Foltz E J, Elkordy M A.
Coronavirus pneumonia following autologous bone marrow
transplantation for breast cancer. Chest 1999; 115: 901-905),
Coronaviruses have been reported to be an important cause of
pneumonia in military recruits, accounting for up to 30% of cases
in some studies (Wenzel R P, Hendley J O, Davies J A, Gwaltney J M,
Coronavirus infections in military recruits: Three-year study with
coronavirus strains OC43 and 229E. Am Rev Respir Dis. 1974; 109:
621-624). Human coronaviruses can infect neurons and viral RNA has
been detected in the brain of patients with multiple sclerosis
(Talbot P J, Cote G, Arbour N. Human coronavirus OC43 and 229E
persistence in neural cell cultures and human brains. Adv Exp Med
Biol.--in press). On the other hand, a number of animal
coronaviruses (eg. Porcine Transmissible Gastroenteritis Virus,
Murine Hepatitis Virus, Avian Infectious Bronchititis Virus) cause
respiratory, gastrointestinal, neurological or hepatic disease in
their respective hosts (McIntosh K. Coronaviruses: a comparative
review. Current Top Microbiol Immunol. 1974; 63: 85-112).
[0334] We describe for the first time the clinical presentation and
complications of SARS. Less than 25% of patients with coronaviral
pneumonia had upper respiratory tract symptoms. As expected in
atypical pneumonia, both respiratory symptoms and positive
auscultatory findings were very disproportional to the chest
radiographic findings. Gastrointestinal symptoms were present in
10%. It is relevant that the virus RNA is detected in faeces of
some patients and that coronaviruses have been associated with
diarrhoea in animals and humans (Caul E O, Egglestone S I. Further
studies on human enteric coronaviruses Arch Virol. 1977; 54:
107-17). The high incidence of deranged liver function test,
leucopenia, significant lymphopenia, thrombocytopenia and
subsequent evolution into adult respiratory distress syndrome
suggests a severe systemic inflammatory damage induced by this
hSARS virus. Thus immuno-modulation by steroid may be important to
complement the antiviral therapy by ribavirin. In this regard, it
is pertinent that severe human disease associated with the avian
influenza subtype H5N1, another virus that recently crossed from
animals to humans, has also been postulated to have an
immuno-pathological component (Cheung C Y, Poon L L M, Lau A S Y et
al. Induction of proinflammatory cytokines in human macrophages by
influenza A (H5N1) viruses: a mechanism for the unusual severity of
human disease. Lancet 2002; 360: 1831-1837). In common with H5N1
disease, patients with severe SARS are adults, are significantly
more lymphopenic and have parameters of organ dysfunction beyond
the respiratory tract (Table 4) (Yuen K Y, Chan P K S, Peiris J S
M, et al. Clinical features and rapid viral diagnosis of human
disease associated with avian influenza A H5N1 virus. Lancet 1998;
351: 467-471). It is important to note that a window of opportunity
of around 8 days exists from the onset of symptoms to respiratory
failure. Severe complicated cases are strongly associated with both
underlying disease and delayed use of ribavirin and steroid
therapy. Following our clinical experience in the initial cases,
this combination therapy was started very early in subsequent cases
which were largely uncomplicated cases at the time of admission.
The overall mortality at the time of writing is only 2% with this
treatment regimen. There were still 8 out of 19 complicated cases
who had not shown significant response. It is not possible to a
detail analysis of the therapeutic response to this combination
regimen due to the heterogeneous dosing and time of initiation of
therapy.
[0335] Other factors associated with severe disease is acquisition
of the disease through household contact which may be attributed to
a higher dose or duration of viral exposure and the presence of
underlying diseases.
[0336] The clinical description reported here pertains largely to
the more severe cases admitted to hospital. We presently have no
data on the full clinical spectrum of the emerging Coronaviridae
infection in the community or in an out-patient-setting. The
availability of diagnostic tests as described here will help
address these questions. In addition, it will allow questions
pertaining to the period of virus shedding (and communicability)
during convalescence, the presence of virus in other body fluids
and excreta and the presence of virus shedding during the
incubation period, to be addressed.
[0337] The epidemiological data at present appears to indicate that
the virus is spread by droplets or by direct and indirect contact
although airborne spread cannot be ruled out in some instances. The
finding of infectious virus in the respiratory tract supports this
contention. Preliminary evidence also suggests that the virus may
be shed in the feces. However, it is important to note that
detection of viral RNA does not prove that the virus is viable or
transmissible. If viable virus is detectable in the feces, this
would be a potentially additional route of transmission that needs
to be considered. It is relevant to note that a number of animal
coronaviruses are spread via the fecal-oral route (McIntosh K.
Coronaviruses: a comparative review. Current Top Microbiol Immunol.
1974; 63: 85-112).
7. Deposit
[0338] A sample of isolated hSARS virus was deposited with China
Center for Type Culture Collection (CCTCC) at Wuhan University,
Wuhan 430072 in China on Apr. 2, 2003 in accordance with the
Budapest Treaty on the Deposit of Microorganisms, and accorded
accession No. CCTCC-V200303, which is incorporated herein by
reference in its entirety.
8. Market Potential
[0339] The hSARS virus can now be grown on a large scale, which
allows the development of various diagnostic tests as described
hereinabove as well as the development of vaccines and antiviral
agents that are effective in preventing, ameliorating or treating
SARS. Given the severity of the disease and its rapid global
spread, it is highly likely that significant demands for diagnostic
tests, therapies and vaccines to battle against the disease, will
arise on a global scale. In addition, this virus contains genetic
information which is extremely important and valuable for clinical
and scientific research applications.
9. Equivalents
[0340] Those skilled in the art will recognize, or be able to
ascertain many equivalents to the specific embodiments of the
invention described herein using no more than routine
experimentation. Such equivalents are intended to be encompassed by
the following claims.
[0341] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
[0342] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
Sequence CWU 0
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References